Abstract:

Compounds comprising condensed particles having diameters less than 1000
nm, wherein the particles comprise one or more double stranded
ribonucleic acids (dsKNAs) and one or more peptides. The compounds,
compositions and methods are useful for modulating gene expression by RNA
Interference.

Claims:

1-60. (canceled)

61. A composition comprising a double-stranded RNA, a first peptide and a
second peptide, wherein the concentration of the first peptide of the
composition provides an N/P ratio of the first peptide and the dsRNA of
from about 0.2 to about 1, and wherein the concentration of the second
peptide of the composition provides an N/P ratio of the composition from
about 0.5 to about 20, and wherein the dsRNA, the first peptide and the
second peptide form a particle having a diameter of from about 0.5 nm to
about 1000 nm.

62. The composition of claim 61, wherein the first peptide is from 5% to
99% of the mass of the particle.

63. The composition of claim 61, wherein the first peptide is from 5% to
50% of the mass of the particle.

64. The composition of claim 61, wherein the first peptide and the second
peptide are from 50% to 99% of the mass of the particle.

65. The composition of claim 61, wherein the amino acid sequence of the
first peptide and the second peptide independently comprise an amino acid
sequence selected from the group consisting of SEQ ID NOS:28-37, 43,
67-71, and 87-95.

66. The composition of claim 61, wherein the amino acid sequence of the
first peptide and the second peptide independently comprise an amino acid
sequence selected from the group consisting of SEQ ID NO:28, 34, 37, 38,
and 39.

67. The composition of claim 61, wherein the amino acid of the first
peptide comprises the amino acid sequence of SEQ ID NO: 28.

68. The composition of claim 61, wherein the first peptide and/or the
second peptide is pegylated.

69. The composition of claim 61, wherein the particle has a diameter of
from 10 to 300 nanometers.

70. The composition of claim 61, wherein the particle has a diameter of
from 40 to 100 nanometers.

71. The composition of claim 61, wherein the particle is cross-linked.

72. The composition of claim 61, wherein the particle has a zeta potential
magnitude of at least 20 mV.

73. The composition of claim 61, wherein the particle has a zeta potential
magnitude of at least 30 mV

74. The composition of claim 61, wherein the dsRNA is an siNA.

75. A method comprising the steps of:a) associating a first peptide and a
double-stranded RNA (dsRNA), wherein the first peptide and the dsRNA form
a first particle and have an N/P ratio of from about 0.2 to about 1;b)
associating a second peptide with the first particle, wherein the second
peptide adjusts the N/P ratio to about 0.5 to about 20; andwherein the
first peptide, the second peptide, and the dsRNA form a second particle
having diameter of less than 1000 nm.

76. The method of claim 75, wherein the first peptide is from 5% to 99% of
the mass of the second particle.

77. The method of claim 75, wherein the first peptide is from 5% to 50% of
the mass of the second particle.

78. The method of claim 75, wherein the first peptide and the second
peptide are from 50% to 99% of the mass of the second particle.

79. The method of claim 75, wherein the amino acid sequence of the first
peptide and the second peptide independently comprise an amino acid
sequence selected from the group consisting of SEQ ID NOS:28-37, 43,
67-71, and 87-95.

80. The method of claim 75, wherein the amino acid sequence of the first
peptide and the second peptide independently comprise an amino acid
sequence selected from the group consisting of SEQ ID NO:28, 34, 37, 38,
and 39.

81. The method of claim 75, wherein the amino acid of the first peptide
comprises the amino acid sequence of SEQ ID NO: 28.

82. The method of claim 75, wherein the first peptide and/or the second
peptide is pegylated.

83. The method of claim 75, wherein the second particle has a diameter of
from 10 to 300 nanometers.

84. The method of claim 75, wherein the second particle has a diameter of
from 40 to 100 nanometers.

85. The method of claim 75, wherein the second particle has a zeta
potential magnitude of at least 20 mV.

86. The method of claim 75, wherein the second particle has a zeta
potential magnitude of at least 30 mV.

87. The method of claim 75, wherein the second particle is cross-linked.

88. The method of claim 75, wherein the first peptide is mixed or admixed
with the dsRNA.

89. The method of claim 75, wherein the second peptide is mixed or admixed
with the first peptide and the dsRNA.

Description:

FIELD OF THE INVENTION

[0001]This invention relates generally to the fields of RNA Interference,
and delivery of RNA therapeutics. More particularly, this invention
relates to compounds and compositions of peptide ribonucleic acid
condensate particles, and their uses for medicaments and for delivery as
therapeutics. This invention relates generally to methods of using
peptide ribonucleic acid condensate compounds in RNA Interference for
gene-specific inhibition of gene expression in mammals.

BACKGROUND OF THE INVENTION

[0002]RNA Interference (RNAi) refers to methods of sequence-specific
post-transcriptional gene silencing which is mediated by a
double-stranded RNA (dsRNA) called a short interfering RNA (siRNA). See
Fire, et al., Nature 391:806, 1998, and Hamilton, et al., Science
286:950-951, 1999. RNAi is shared by diverse flora and phyla and is
believed to be an evolutionarily-conserved cellular defense mechanism
against the expression of foreign genes. See Fire, et al., Trends Genet.
15:358, 1999.

[0003]RNAi is therefore a ubiquitous, endogenous mechanism that uses small
noncoding RNAs to silence gene expression. See Dykxhoorn, D. M. and J.
Lieberman, Annu. Rev. Biomed. Eng. 8:377-402, 2006. RNAi can regulate
important genes involved in cell death, differentiation, and development.
RNAi may also protect the genome from invading genetic elements, encoded
by transposons and viruses. When a siRNA is introduced into a cell, it
binds to the endogenous RNAi machinery to disrupt the expression of mRNA
containing complementary sequences with high specificity. Any
disease-causing gene and any cell type or tissue can potentially be
targeted. This technique has been rapidly utilized for gene-function
analysis and drug-target discovery and validation. Harnessing RNAi also
holds great promise for therapy, although introducing siRNAs into cells
in vivo remains an important obstacle.

[0004]The mechanism of RNAi, although not yet fully characterized, is
through cleavage of a target mRNA. The RNAi response involves an
endonuclease complex known as the RNA-induced silencing complex (RISC),
which mediates cleavage of a single-stranded RNA complementary to the
antisense strand of the siRNA duplex. Cleavage of the target RNA takes
place in the middle of the region complementary to the antisense strand
of the siRNA duplex (Elbashir, et al., Genes Dev. 15:188, 2001).

[0005]One way to carry out RNAi is to introduce or express a siRNA in
cells. Another way is to make use of an endogenous ribonuclease III
enzyme called dicer. One activity of dicer is to process a long dsRNA
into siRNAs. See Hamilton, et al., Science 286:950-951, 1999; Berstein,
et al., Nature 409:363, 2001. A siRNA derived from dicer is typically
about 21-23 nucleotides in overall length with about 19 base pairs
duplexed. See Hamilton, et al., supra; Elbashir, et al., Genes Dev.
15:188, 2001. In essence, a long dsRNA can be introduced in a cell as a
precursor of a siRNA.

[0006]The development of RNAi therapy, antisense therapy, and gene
therapy, among others, has created a need for effective means of
introducing active nucleic acid-based agents into cells. In general,
nucleic acids are stable for only very limited times in cells or plasma.
However, nucleic acid-based agents can be stabilized by aggregation and
binding into condensed compounds which may exhibit particles small enough
for cellular delivery.

[0007]What is need are compounds comprised of small particles which
contain an active nucleic acid agent for intracellular delivery and,
ultimately, as a therapeutic, and methods for making such compounds. In
particular, there is a need for compounds and methods to deliver
double-stranded RNA to cells to produce the response of RNAi.

SUMMARY OF THE INVENTION

[0008]This invention overcomes these and other drawbacks in the field by
providing a range of peptide-ribonucleic acid compounds and compositions
for use in RNA Interference and other therapeutic methods. This invention
particularly provides compounds and methods of making compounds
comprising one or more ribonucleic acid agents condensed with one or more
peptides into small stable particles which are active to inhibit
expression of targeted genes through RNA Interference. This summary,
taken along with the description of drawings, detailed description of the
invention, as well as the appended examples, claims, and drawings, as a
whole, encompasses the invention disclosed.

[0009]In some aspects, this invention provides a range of peptide-RNA
compounds and compositions for use in RNA Interference and other
therapeutic methods, including compounds containing RNAs and peptides
condensed into small, stable particles, which are active to inhibit
expression of targeted genes through RNAi. The compounds of this
invention are generally provided as a wide range of admixtures or
condensates of synthetic peptides with nucleic acids.

[0010]In other aspects, the condensate compounds and compositions of this
invention include small, stable particles of a peptide-RNA complex. In
some embodiments, these compounds and particles can be further stabilized
by crosslinking. In other embodiments, the compounds and compositions of
this invention include a stealthing or surface modifying agent such as
polyethylene glycol to enhance delivery.

[0011]In further aspects, the compounds of this invention include
condensate complexes of one or more ribonucleic acids and one or more
peptide components. The peptide components can have sufficient positive
charge to bind to a ribonucleic acid to form a non-covalently linked
peptide-ribonucleic acid condensate compound.

[0012]In some aspects, condensate compounds of this invention may form
uniform particles. In some embodiments, the diameters of spherical
particles of peptide-nucleic acid compounds may have a narrow
distribution with an average of less than 1000 nanometers (nm).

[0013]The peptide-nucleic acid condensate compounds of this invention can
provide their own multicomponent formulations. In some embodiments, a
compound can be combined with other agents for drug delivery such as
carriers or vehicles for delivery to a cell, or various delivery
matrices, for in vivo therapeutics.

[0014]In some embodiments, compounds are provided from one or more
ribonucleic acids and one or more peptides by dissolving at least one
ribonucleic acid agent in an aqueous solution, then adding at least one
peptide component to the aqueous solution thereby condensing particles
having diameters less than 1000 nm, thereafter adding a second or
successive peptide components to the aqueous solution, which adds mass to
the particles.

[0015]In further embodiments, compounds are provided from one or more
ribonucleic acid agents and one or more peptide components by dissolving
a first peptide component in an aqueous solution, then adding the
ribonucleic acid agent to the aqueous solution thereby condensing
particles having diameters less than 1000 nm, thereafter adding a second
or successive peptide components to the aqueous solution, which adds mass
to the particles.

[0016]In one aspect of this invention, a peptide component is selected by
its relative affinity for a nucleic acid. The peptide components can be
selected to allow a variation of the degree of binding of the peptide
components to the nucleic acids.

[0017]In some aspects, ribonucleic acid-peptide condensate compounds can
be reversibly-bound. Compounds of ribonucleic acids and an amount of
positively-charged ribonucleic acid-binding peptides can be substantially
stable in an extracellular biological environment and release ribonucleic
acids upon contact with an intracellular endosome. The release may
produce the response of RNAi.

[0018]In further aspects, structures and methods of stabilizing a
peptide-ribonucleic acid compound are provided including crosslinking
ribonucleic acid-binding peptides within the compound. Methods of
protecting a peptide-ribonucleic acid compound from degradation within a
biological organism include crosslinking at least a portion of the
peptides within the compound.

[0019]This invention further provides uses of the compounds as medicaments
and in the manufacture of medicaments for use in RNAi therapy in animals
and humans.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020]FIG. 1: Diameters of condensate particles of siRNA G1498 and peptide
PN183 at various concentrations of G1498 and various nitrogen to
phosphorous ratios (N/P). For each group of three bars at a particular
N/P, the concentration of G1498 for the leftmost bar was 100 ug/ml, for
the middle bar was 50 ug/ml, and for the rightmost bar was 10 ug/ml. At
N/P of 0.2 and 0.5, the particles were very small when the concentration
of G1498 was 10 ug/ml.

[0021]FIG. 2: Diameters of condensate particles of siRNA G1498 and peptide
PN183 at various nitrogen to phosphorous ratios (N/P). For each group of
two bars at a particular N/P, the left bar was with vortexing, while the
right bar was without vortexing. Data obtained immediately after mixing.

[0022]FIG. 3: Diameters of condensate particles of siRNA G1498 and peptide
PN183 at various nitrogen to phosphorous ratios (N/P). For each group of
two bars at a particular N/P, the left bar was with vortexing, while the
right bar was without vortexing. Data obtained 30 minutes after mixing.

[0023]FIG. 4: Diameters of condensate particles of siRNA G1498 and peptide
PN183 at various nitrogen to phosphorous ratios (N/P). For each group of
two bars at a particular N/P, the left bar was with vortexing, while the
right bar was without vortexing. Data obtained 60 minutes after mixing.

[0024]FIG. 5: Diameters of condensate particles of siRNA G1498 and peptide
PN183 at various nitrogen to phosphorous ratios (N/P). For each group of
two bars at a particular N/P, the left bar was with vortexing, while the
right bar was without vortexing. Data obtained 24 hours after mixing.

[0025]FIG. 6: Diameters of condensate particles of siRNA G1498 and peptide
PN183 obtained at a concentration of G1498 of 100 ug/ml for various
values of pH.

[0026]FIG. 7: Diameters of condensate particles of siRNA G1498 and peptide
PN183 obtained as the concentration of sodium chloride was increased.

[0027]FIG. 8: Diameters of condensate particles of siRNA G1498 and peptide
PN183 obtained at various N/P ratios and various order of addition of the
components. For each group of two bars at a particular N/P, the left bar
was obtained by adding siRNA first, while the right bar was obtained by
adding the peptide first.

[0030]FIG. 11: Knockdown assay of LPS-induced TFN-α expression
(pg/ml) in a mouse model by intranasal administration of a composition
including condensate particles of siRNA Inm-4 and peptides PN183 and
PN939. Buffer control is the leftmost bar, followed by data for
condensate Inm-4/PN183/PN939, followed on the right by data for compound
Inm-4/PN183/PN939 crosslinked with glutaraldehyde (G). Placebo does not
contain the siRNA, and Qneg contains a non-active-siRNA.

[0031]FIG. 12: Knockdown in vitro assay of lac-z expression in rat
gliosarcoma fibroblast cells 9L/LacZ for condensates of the lac-z siRNA
with peptide PN183 and various second peptides. Comparative data using
HiPerFect® (Qiagen; Valencia, Calif.) is the leftmost bar, followed by
data for various compounds of this invention. The N/P ratio for PN183 was
0.75, while the N/P ratio for the second peptide was 0.3.

DETAILED DESCRIPTION OF INVENTION

[0032]This invention provides a range of peptide-RNA compounds and
compositions for use in RNA Interference and other therapeutic methods.
More particularly, this invention includes compounds containing RNA and
peptide condensed into small, stable particles, which are active to
inhibit expression of targeted genes through RNAi.

[0033]The compounds of this invention are generally provided as admixtures
or condensates of synthetic peptides with nucleic acids. A wide range of
peptides may be used to form the compounds. The mass of a peptide is
typically less than about 120 kDa, or less than about 60 kDa, or less
than about 30 kDa. A peptide of the compound may be a mucosal
permeability modulator or mucosal permeation enhancer.

[0034]The condensate compounds include small, stable particles of a
peptide-RNA complex. These compounds and particles can be further
stabilized by crosslinking with various reagents. In some embodiments,
the compounds and compositions of this invention include a stealthing or
surface modifying agent such as polyethylene glycol to enhance delivery.

[0035]The compounds of this invention include condensate complexes
comprised of one or more ribonucleic acids and one or more peptide
components. The peptide components can have sufficient positive charge to
bind to a ribonucleic acid to form a non-covalently linked
peptide-ribonucleic acid condensate compound. Stable ribonucleic acid
complexes are provided which are comprised of ribonucleic acids and an
amount of ribonucleic acid-binding peptides effective to stabilize the
ribonucleic acid under in vivo conditions. The binding of the components
of the peptide-nucleic acid complex is due partly to ionic forces, and
can involve various other interactions such as van der Waals forces or
hydrogen bonding.

[0036]Peptide-nucleic acid condensate compounds of this invention can
comprise uniform particles. The diameters of spherical particles of the
peptide-nucleic acid compounds may have a narrow distribution with an
average of less than 1000 nanometers (nm). The diameters of spherical
particles may be less than 1000 nanometers, from about 0.5 to about 400
nanometers, from about 10 to about 300 nanometers, and from about 40 to
about 100 nanometers. The magnitude of the zeta potential for stable
particles can be greater than about 20 millivolts, or greater than about
30 millivolts.

[0037]As used herein, the term "uniform" means that a substantial portion
of the particles of a compound have a narrow distribution of diameters.
More than one distribution of diameters may occur in a compound of
uniform particles. A narrow distribution of diameters corresponds to a
peak in the particle size distribution chart which is based on the raw
correlation coefficient versus time data of a particle sizer instrument.
Preferably, a uniform compound has at least 30% of the particles in one
narrow distribution of diameters.

[0038]The peptide-nucleic acid condensate compounds of this invention
provide their own multicomponent formulations, and can be further
combined with other agents for drug delivery such as carriers or vehicles
for delivery to a cell, or various delivery matrices, for in vivo
therapeutics.

[0039]The compound and compositions of this invention may be dispersed
within a pharmaceutically acceptable medium, associated with a matrix, or
associated with a carrier or vehicle for delivery to a cell or subject. A
solution comprised of a dispersion of the compounds or particles of this
invention can be provided for delivery as a therapeutic.

Peptide Components

[0040]Peptide components suitable for the compounds of this invention may
be synthetically or derived from natural or other sources.

[0041]The peptide components can contain from 2 to about 1000 amino acids
in length; from 2 to about 600 amino acids in length; from 2 to about 60
amino acids in length; from 5 to about 30 amino acids in length; and from
5 to about 25 amino acids in length.

[0042]The peptide components may comprise a plurality of positive charges.
For example, a peptide component may comprise from 1 to about 100
positive charges, from 5 to about 30 positive charges, and from 9 to
about 15 positive charges. The positive charges of a peptide component
can be provided by positively-charged lysine or arginine residues.

[0043]A wide range of peptides may be used to form the peptide-nucleic
acids compounds. The mass of a peptide component is typically less than
about 120 kDa, or less than about 60 kDa, or less than about 30 kDa. The
peptide of the peptide component may optionally be conjugated, or
derivatized with a polymer such as a polyalkyleneoxide,
polyethyleneoxide, polypropyleneoxide, or combinations thereof. For
example, the peptide components of the compounds of this invention may be
covalently derivatized with polyethyleneglycol (PEG).

[0044]Functional domains of the polynucleotide delivery-enhancing
polypeptides are useful for the ability to deliver siNAs into cells.
These functional domains include membrane attachment, fusogenic and
nucleotide binding regions. Membrane attachment describes the ability of
the exemplary polynucleotide delivery-enhancing polypeptide to bind the
cell membrane. The fusogenic character reflects an ability to detach from
the cell membrane and enter the cytoplasm. The membrane attachment and
fusogenic domains of the peptide are closely linked mechanistically
(i.e., peptide's ability to enter the cell) and therefore may be
difficult to differentiate experimentally. Lastly, the nucleotide binding
describes the peptide's ability to bind nucleotides.

[0045]A peptide of the compound may contain structural features which are
known to enhance delivery of a compound across a barrier, such as a
mucosal barrier. Examples of delivery enhancing features include various
protein transduction domains. A peptide component can be a mucosal
permeability modulator.

[0046]Examples of protein transduction domains for polynucleotide
delivery-enhancing polypeptides of the invention include:

[0050]Table 1 demonstrates a conservative zinc finger motif for double
strand DNA binding which is characterized by the
C-x(2,4)-C-x(12)-H-x(3)-H motif pattern (SEQ ID NO: 27), which itself can
be used to select and design additional polynucleotide delivery-enhancing
polypeptides according to the invention.

[0052]In some embodiments of this invention, polynucleotide
delivery-enhancing polypeptides may be constructed by combining any of
the foregoing structural elements, domains, or motifs into a single
polypeptide which mediates enhanced delivery of siNAs into target cells.
For example, a protein transduction domain of the TAT polypeptide may be
fused to the N-terminal 20 amino acids of the influenza virus
hemagglutinin protein, termed HA2, to yield a polynucleotide
delivery-enhancing polypeptide.

[0053]The compounds of this invention can include one or more peptide
components. A peptide component can have sufficient positive charge to
bind a ribonucleic acid to form a non-covalently bound
peptide-ribonucleic acid condensate compound. While the binding of the
components of the peptide-nucleic acid complex is due partly to ionic
forces, the binding can also involve various other interactions such as
van der Waals forces, hydrogen bonding, or hydrophobic interactions. A
complex may retain aqueous interactions, or a region of high solvent
concentration.

[0054]Stable peptide-ribonucleic acid complexes are provided which
comprise ribonucleic acids and an amount of ribonucleic acid-binding
peptides effective to stabilize the ribonucleic acid under in vivo
conditions.

[0055]Some example peptides useful for compounds of this invention are
shown in Table 2.

[0056]Further examples of peptides useful for compounds of this invention
are given in the examples appended below.

Condensate Compounds and their Preparation

[0057]This invention provides peptide-ribonucleic acid condensate
compounds which can be comprised of particles having diameters less than
about 1000 nm, from about 0.5 nm to about 400 nm; from about 10 nm to
about 300 nm; and from about 40 nm to about 100 nm.

[0058]The peptide components of the compounds may be from 5-95% of the
mass of the particles, or from 45-95% of the mass of the particles.

[0059]In some embodiments of this invention, peptide-nucleic acid
compounds are provided from one or more ribonucleic acid agents and one
or more peptide components by condensing the ribonucleic acid agents with
the peptide components in an aqueous solution, thereby forming particles
having diameters less than 1000 nm.

[0060]In general, the compounds of this invention comprise peptide-nucleic
acid condensates having been formed from one or more peptides and one or
more nucleic acids. The condensates are characterized in part by the
nitrogen to phosphorous ratio (N/P ratio) for the peptides in relation to
the nucleic acids.

[0061]A compound of this invention may be comprised of condensed particles
having diameters less than 1000 nm, wherein each particle comprises at
least 10 double stranded ribonucleic acid (dsRNA) molecules and at least
10 peptides. As used herein, "at least 10 peptides" refers to a partial
molar quantity being 10 peptide molecules, which may be the same or
different in structure. Thus, "at least 10 peptides" can be a partial
molar quantity of a single peptide structure, or partial molar quantities
of two or more different peptide structures.

[0062]In general, as used herein, terms such as "peptide" and "nucleic
acid" and "dsRNA" and "siRNA" refer to an amount of those molecules
sufficient to form a compound of this invention. In other words, in
general, such terms refer to partial molar quantities rather than
individual molecules. A "peptide" is one or more peptide molecules such
as, for example, Avagadro's number of peptide molecules. "Adding two
peptides to a ribonucleic acid agent" refers to an admixture of peptides
of two different structures, each in partial molar quantity, to the
ribonucleic acid agent.

[0063]The amount of peptide bound to the nucleic acids (NAs) in a complex
or condensate can be obtained from the amount of bound nucleic acids
using the peptide:NA charge ratio for single molecule pairing, also
called the nitrogen to phosphorous ratio (N/P ratio). The amount of free
peptide remaining in solution after condensation is given by mass
balance. Thus, the charge ratio N/P herein refers to the initial charge
ratio N/P of a single peptide component to a single nucleic acid agent in
the initial condensate solution.

[0064]In general, the concentration of the nucleic acid agents in the
solution is limited only by their solubility. The concentrations of the
peptide components of the solution are adjusted to provide a desired N/P
ratio.

[0065]In some embodiments, the concentrations of the peptide components of
the solution are adjusted to provide a combined N/P ratio of about one.
When the N/P ratio is about one, then on the basis of ionic charge
neither the peptide components nor the nucleic acid agents are in excess.

[0066]In some embodiments, the concentration of each peptide component of
the solution is adjusted to provide an N/P ratio of from about 0.2 to
about 50, from about 0.5 to about 20, from about 0.5 to about 7, or from
about 0.5 to about 2.5.

[0067]The pH of the solution is typically less than about 11, less than
about 9, and less than about 8. The solution can optionally be vortexed
for mixing the components.

[0068]In some embodiments, the condensate compounds are prepared by adding
nucleic acid agents to a solution containing the peptide components.

[0069]In some embodiments, the solution may contain an inorganic or
organic salt. For example, the aqueous solution may contain sodium
chloride at a concentration of less than or equal to about 1 M, less than
or equal to about 0.5 M, and less than or equal to about 0.25 M.

[0070]Optionally, the peptide-nucleic acid condensate compounds of a
particular distribution of sizes can be isolated from the solution. In
some embodiments, the solution containing the peptide-nucleic acid
condensate compounds is filtered to isolate particles of various sizes.

[0071]In other embodiments, the solution containing the peptide-nucleic
acid condensate compounds is dialyzed to remove excess or unbound peptide
components.

[0073]In some embodiments of this invention, peptide-nucleic acid
compounds are provided from one or more ribonucleic acid agents and one
or more peptide components by dissolving at least one ribonucleic acid
agent in an aqueous solution, then adding at least one peptide component
to the aqueous solution thereby condensing particles having diameters
less than 1000 nm, thereafter adding a second or successive peptide
components to the aqueous solution, thereby adding mass to the particles.

[0074]In some embodiments of this invention, peptide-nucleic acid
compounds are provided from one or more ribonucleic acid agents and one
or more peptide components by dissolving a first peptide component in an
aqueous solution, then adding the ribonucleic acid agent to the aqueous
solution thereby condensing particles having diameters less than 1000 nm,
thereafter adding a second or successive peptide components to the
aqueous solution, thereby adding mass to the particles.

[0075]In one aspect of this invention, peptide-nucleic acid compounds are
provided in which a peptide component is selected by its relative
affinity for the nucleic acid. For example, a relative binding analysis
of various peptides to a nucleic acid is performed by measurement of the
displacement of SYBR-gold nucleic acid binding dye by the peptide. By
characterizing the relative affinity of the peptide components for the
nucleic acids of the compounds, the peptide components can be selected to
allow a variation of the degree of binding of the peptide components to
the nucleic acids.

[0076]Varying the degree of binding of the peptide components to the
nucleic acids allows the condensate particles to be formed with a
stronger-binding peptide component first, followed by a weaker-binding
peptide component, or vice-versa, or to have multiple additions of
components of variable binding strength.

[0077]In some embodiments, it is desirable to have the first peptide
component which is condensed with the nucleic acid agent to have a higher
binding affinity for the nucleic acid agent than succeeding peptide
components. In these embodiments the concentration of the first peptide
component of the solution is adjusted to provide an N/P ratio of from
about 0.2 to about 7, from about 0.2 to about 2.5, or from about 0.2 to
about 1. In these embodiments the concentrations of succeeding peptide
components is adjusted to provide an NIP ratio of from about 0.2 to about
50, from about 0.5 to about 20, from about 0.5 to about 7, or from about
0.5 to about 2.5.

[0078]Reversibly-bound ribonucleic acid-peptide condensate compounds
comprise ribonucleic acids and an amount of positively-charged
ribonucleic acid-binding peptides that form a ribonucleic acid-peptide
condensate that is substantially stable in an extracellular biological
environment and that can release ribonucleic acids upon contact with an
intracellular endosome.

[0079]A population of peptide-nucleic acid condensates is provided in
which the peptides comprise an amount of positively-charged residues
effective to bind ribonucleic acids. The ribonucleic acid-peptide
condensates are substantially stable in an extracellular biological
environment and can release ribonucleic acids intracellularly in a manner
effective to produce the response of RNAi.

[0080]In some aspects of this invention, reagents are used to crosslink
the peptide-RNA condensates. For example, the stability of peptide-RNA
condensates may be increased by introducing dialdehyde groups, such as
glutaraldehyde, to crosslink surface amine groups on the peptides or
particles. Other examples of crosslinkers include formaldehyde, acrolein,
and dithiobis(succinimidylpropionate). Crosslinked condensate compounds
may have improved resistance to metabolism by serum endonucleases.

[0081]In some embodiments, a first peptide component which is condensed
with the nucleic acid agent is crosslinked before the addition of
successive peptide components. Optionally, the condensate of a first
peptide component can be crosslinked after the addition of successive
peptide components. In some embodiments, the condensate of a first
peptide component is crosslinked before and after the addition of
successive peptide components.

[0082]Methods of stabilizing a peptide-ribonucleic acid compound include
crosslinking ribonucleic acid-binding peptides within the compound with,
for example, a glutaraldehyde crosslinker. Methods of protecting a
peptide-ribonucleic acid compound from degradation within a biological
organism include crosslinking at least a portion of the peptides within
the compound using, for example, a glutaraldehyde crosslinker.

[0083]The peptide-ribonucleic acid compounds of this invention can also be
stabilized by addition of surface modifying agents such as surfactants,
neutral lipids, or a polyethyleneoxide. For example, polyethylene glycol
added to a solution of the condensate compounds can adhere to the
particles thereof. A nonionic polyoxyethylene-polyoxypropylene block
co-polymer may be added, for example, to stabilize the particles of the
compound.

[0084]Uses of the compounds of this invention in the manufacture of
medicaments for use in RNAi therapy in animals and humans are encompassed
herein.

[0086]In this context, this invention provides compounds, compositions and
methods for modulating gene expression by RNA Interference. A compound or
composition of this invention may release a ribonucleic acid agent to a
cell which can produce the response of RNAi. Compounds or compositions of
this invention may release ribonucleic acid agents to a cell upon contact
with an intracellular endosome. The release of a ribonucleic acid agent
intracellularly may provide inhibition of gene expression in the cell.

[0087]Ribonucleic acid agents useful for this invention may be targeted to
various genes. For example, a siRNA agent of this invention may have a
sequence that is complementary to a region of a TNF-alpha gene. In some
embodiments of this invention, compounds and compositions are useful to
regulate expression of tumor necrosis factor-α (TNF-α).
TNF-α can be linked, for example, to inflammatory processes which
occur in pulmonary diseases, and can have anti-inflammatory effects.
Blocking TNF-α by delivery of a composition of this invention can
be useful to treat or prevent the signs and/or symptoms of rheumatoid
arthritis.

[0089]Expression and/or activity of TNF-α can be modulated by
delivering to a cell, for example, the siRNA molecule Inm-4. Inm-4 is a
double stranded 21-nt siRNA molecule with sequence homology to the human
TNF-α gene. Inm-4 has a 3' dTdT overhang on the sense strand and a
3' dAdT overhang on the antisense strand. The primary structure of Inm-4
is

[0090]Expression and/or activity of TNF-α can be modulated by
delivering to a cell, for example, the siRNA molecule LC20. LC20 is a
double stranded 21-nt siRNA molecule with sequence homology to the human
TNF-α gene. LC20 is directed against the 3'-UTR region of human
TNF-α. LC20 has 19 base pairs with a 3' dTdT overhang on the sense
strand and a 3' dAdT overhang on the antisense strand. The molecular
weight of the sodium salt form is 14,298. The primary structure of LC20
is

[0091]A siRNA of this invention may have a sequence that is complementary
to a region of a viral gene. For example, some compositions and methods
of this invention are useful to regulate expression of the viral genome
of an influenza.

[0092]In this context, this invention provides compositions and methods
for modulating expression and infectious activity of an influenza by RNA
Interference. Expression and/or activity of an influenza can be modulated
by delivering to a cell, for example, a short interfering RNA molecule
having a sequence that is complementary to a region of a RNA polymerase
subunit of an influenza. For example, in Table 3 are shown
double-stranded siRNA molecules with sequence homology to an RNA
polymerase subunit of an influenza.

[0093]A siRNA of this invention may have a sequence that is complementary
to a region of a RNA polymerase subunit of an influenza.

[0094]This invention provides compositions and methods to administer siNAs
directed against a mRNA of an influenza, which effectively down-regulates
an influenza RNA and thereby reduces, prevents, or ameliorates an
influenza infection.

RNA Interference Therapeutics

[0095]In some embodiments, this invention provides compounds, compositions
and methods for inhibiting expression of a target transcript in a subject
by administering to the subject a composition containing an effective
amount of an RNAi-inducing compound such as a short interfering
oligonucleotide molecule, or a precursor thereof. RNAi uses small
interfering RNAs (siRNAs) to target messenger RNA (mRNAs) and attenuate
translation. A siRNA as used in this invention may be a precursor for
dicer processing such as, for example, a long dsRNA processed into a
siRNA. This invention provides methods of treating or preventing diseases
or conditions associated with expression of a target transcript or
activity of a peptide or protein encoded by the target transcript.

[0096]A therapeutic strategy based on RNAi can be used to treat a wide
range of diseases by shutting down the growth or function of a virus or
microorganism, as well as by shutting down the function of an endogenous
gene product in the pathway of the disease.

[0097]In some embodiments, this invention provides novel compositions and
methods for delivery of RNAi-inducing compounds such as short interfering
oligonucleotide molecules, and precursors thereof. In particular, this
invention provides compositions containing an RNAi-inducing compound
which is targeted to one or more transcripts of a cell, tissue, and/or
organ of a subject.

[0098]A siRNA can be two RNA strands having a region of complementarity
about 19 nucleotides in length. A siRNA optionally includes one or two
single-stranded overhangs or loops.

[0099]A shRNA can be a single RNA strand having a region of
self-complementarity. The single RNA strand may form a hairpin structure
with a stem and loop and, optionally, one or more unpaired portions at
the 5' and/or 3' portion of the RNA.

[0101]A siRNA agent of this invention may have a sequence that is
complementary to a region of a target gene. A siRNA of this invention may
have 29-50 base pairs, for example, a dsRNA having a sequence that is
complementary to a region of a target gene. Alternately, the
double-stranded nucleic acid can be a dsDNA.

[0102]In some embodiments, the active agent can be a short interfering
nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA
(dsRNA), micro-RNA, or short hairpin RNA (shRNA) that can modulate
expression of a gene product.

[0103]Comparable methods and compositions are provided that target
expression of one or more different genes associated with a particular
disease condition in a subject, including any of a large number of genes
whose expression is known to be aberrantly increased as a causal or
contributing factor associated with the selected disease condition.

[0104]The RNAi-inducing compound of this invention can be administered in
conjunction with other known treatments for a disease condition.

[0105]In some embodiments, this invention features compositions containing
a small nucleic acid molecule, such as short interfering nucleic acid, a
short interfering RNA, a double-stranded RNA, a micro-RNA, or a short
hairpin RNA, admixed or complexed with, or conjugated to, a
delivery-enhancing compound.

[0107]In some embodiments, the siNA is a double-stranded polynucleotide
molecule comprising self-complementary sense and antisense regions,
wherein the antisense region comprises a nucleotide sequence that is
complementary to a nucleotide sequence in a target ribonucleic acid
molecule for down regulating expression, or a portion thereof, and the
sense region comprises a nucleotide sequence corresponding to (i.e.,
which is substantially identical in sequence to) the target ribonucleic
acid sequence or portion thereof.

[0108]"siNA" means a small interfering nucleic acid, for example a siRNA,
that is a short-length double-stranded nucleic acid, or optionally a
longer precursor thereof. The length of useful siNAs within this
invention will in some embodiments be optimized at a length of
approximately 20 to 50 bp. However, there is no particular limitation to
the length of useful siNAs, including siRNAs. For example, siNAs can
initially be presented to cells in a precursor form that is substantially
different than a final or processed form of the siNA that will exist and
exert gene silencing activity upon delivery, or after delivery, to the
target cell. Precursor forms of siNAs may, for example, include precursor
sequence elements that are processed, degraded, altered, or cleaved at or
after the time of delivery to yield a siNA that is active within the cell
to mediate gene silencing. In some embodiments, useful siNAs will have a
precursor length, for example, of approximately 100-200 base pairs, or
50-100 base pairs, or less than about 50 base pairs, which will yield an
active, processed siNA within the target cell. In other embodiments, a
useful siNA or siNA precursor will be approximately 10 to 49 bp, or 15 to
35 bp, or about 21 to 30 bp in length.

[0109]In some embodiments of this invention, polynucleotide
delivery-enhancing polypeptides are used to facilitate delivery of larger
nucleic acid molecules than conventional siNAs, including large nucleic
acid precursors of siNAs. For example, the methods and compositions
herein may be employed for enhancing delivery of larger nucleic acids
that represent "precursors" to desired siNAs, wherein the precursor amino
acids may be cleaved or otherwise processed before, during or after
delivery to a target cell to form an active siNA for modulating gene
expression within the target cell.

[0110]For example, a siNA precursor polynucleotide may be selected as a
circular, single-stranded polynucleotide, having two or more loop
structures and a stem comprising self-complementary sense and antisense
regions, wherein the antisense region comprises a nucleotide sequence
that is complementary to a nucleotide sequence in a target nucleic acid
molecule or a portion thereof, and the sense region having nucleotide
sequence corresponding to the target nucleic acid sequence or a portion
thereof, and wherein the circular polynucleotide can be processed either
in vivo or in vitro to generate an active siNA molecule capable of
mediating RNAi.

[0111]siNA molecules of this invention, particularly non-precursor forms,
can be less than 30 base pairs, or about 17-19 bp, or 19-21 bp, or 21-23
bp.

[0112]siRNAs can mediate selective gene silencing in the mammalian system.
Hairpin RNAs, with a short loop and 19 to 27 base pairs in the stem, also
selectively silence expression of genes that are homologous to the
sequence in the double-stranded stem. Mammalian cells can convert short
hairpin RNA into siRNA to mediate selective gene silencing.

[0113]RISC mediates cleavage of single stranded RNA having sequence
complementary to the antisense strand of the siRNA duplex. Cleavage of
the target RNA takes place within the region complementary to the
antisense strand of the siRNA duplex. siRNA duplexes of 21 nucleotides
are typically most active when containing two-nucleotide 3'-overhangs.

[0114]Replacing the 3'-overhanging segments of a 21-mer siRNA duplex
having 2-nucleotide 3' overhangs with deoxyribonucleotides may not have
an adverse effect on RNAi activity. Replacing up to 4 nucleotides on each
end of the siRNA with deoxyribonucleotides can be tolerated whereas
complete substitution with deoxyribonucleotides may result in no RNAi
activity.

[0115]Alternatively, the siNAs can be delivered as single or multiple
transcription products expressed by a polynucleotide vector encoding the
single or multiple siNAs and directing their expression within target
cells. In these embodiments the double-stranded portion of a final
transcription product of the siRNAs to be expressed within the target
cell can be, for example, 15 to 49 bp, 15 to 35 bp, or about 21 to 30 by
long.

[0116]In some embodiments of this invention, the double-stranded region of
siNAs in which two strands are paired may contain bulge or mismatched
portions, or both. Double-stranded portions of siNAs in which two strands
are paired are not limited to completely paired nucleotide segments, and
may contain nonpairing portions due to, for example, mismatch (the
corresponding nucleotides not being complementary), bulge (lacking in the
corresponding complementary nucleotide on one strand), or overhang.
Nonpairing portions can be contained to the extent that they do not
interfere with siNA formation. In some embodiments, a "bulge" may
comprise 1 to 2 nonpairing nucleotides, and the double-stranded region of
siNAs in which two strands pair up may contain from about 1 to 7, or
about 1 to 5 bulges. In addition, "mismatch" portions contained in the
double-stranded region of siNAs may be present in numbers from about 1 to
7, or about 1 to 5. Most often in the case of mismatches, one of the
nucleotides is guanine, and the other is uracil. Such mismatching may be
attributable, for example, to a mutation from C to T, G to A, or mixtures
thereof, in a corresponding DNA coding for sense RNA, but other causes
are also contemplated.

[0117]The terminal structure of siNAs of this invention may be either
blunt or cohesive (overhanging) as long as the siNA retains its activity
to silence expression of target genes. The cohesive (overhanging) end
structure is not limited to the 3' overhang, but includes the 5'
overhanging structure as long as it retains activity for inducing gene
silencing. In addition, the number of overhanging nucleotides is not
limited to 2 or 3 nucleotides, but can be any number of nucleotides as
long as it retains activity for inducing gene silencing. For example,
overhangs may comprise from 1 to about 8 nucleotides, or from 2 to 4
nucleotides.

[0118]The length of siNAs having cohesive (overhanging) end structure may
be expressed in terms of the paired duplex portion and any overhanging
portion at each end. For example, a 25/27-mer siNA duplex with a 2-bp 3'
antisense overhang has a 25-mer sense strand and a 27-mer antisense
strand, where the paired portion has a length of 25 bp.

[0119]Any overhang sequence may have low specificity to a target gene, and
may not be complementary (antisense) or identical (sense) to the target
gene sequence. As long as the siNA retains activity for gene silencing,
it may contain in the overhang portion a low molecular weight structure,
for example, a natural RNA molecule such as a tRNA, an rRNA, a viral RNA,
or an artificial RNA molecule.

[0120]The terminal structure of the siNAs may have a stem-loop structure
in which ends of one side of the double-stranded nucleic acid are
connected by a linker nucleic acid, e.g., a linker RNA. The length of the
double-stranded region (stem-loop portion) can be, for example, 15 to 49
bp, or 15 to 35 bp, or about 21 to 30 by long. Alternatively, the length
of the double-stranded) region that is a final transcription product of
siNAs to be expressed in a target cell may be, for example, approximately
15 to 49 bp, or 15 to 35 bp, or about 21 to 30 by long.

[0121]The siNA can contain a single stranded polynucleotide having a
nucleotide sequence complementary to a nucleotide sequence in a target
nucleic acid molecule, or a portion thereof, wherein the single stranded
polynucleotide can contain a terminal phosphate group, such as a
5'-phosphate (see for example, Martinez, et al., Cell. 110:563-574, 2002,
and Schwarz, et al., Molecular Cell 10:537-568, 2002, or
5',3'-diphosphate.

[0122]As used herein, the term siNA molecule is not limited to molecules
containing only naturally-occurring RNA or DNA, but also encompasses
chemically-modified nucleotides and non-nucleotides. In some embodiments,
the short interfering nucleic acid molecules of the invention lack
2'-hydroxy (2'-OH) containing nucleotides. In some embodiments, short
interfering nucleic acids do not require the presence of nucleotides
having a 2'-hydroxy group for mediating RNAi and as such, short
interfering nucleic acid molecules of this invention optionally do not
include any ribonucleotides (e.g., nucleotides having a 2'-OH group).
siNA molecules that do not require the presence of ribonucleotides within
the siNA molecule to support RNAi can, however, have an attached linker
or linkers or other attached or associated groups, moieties, or chains
containing one or more nucleotides with 2'-OH groups. siNA molecules can
comprise ribonucleotides in at least about 5, 10, 20, 30, 40, or 50% of
the nucleotide positions.

[0125]"Antisense RNA" is an RNA strand having a sequence complementary to
a target gene mRNA, that can induce RNAi by binding to the target gene
mRNA.

[0126]"Sense RNA" is an RNA strand having a sequence complementary to an
antisense RNA, and anneals to its complementary antisense RNA to form a
siRNA.

[0127]As used herein, the term "RNAi construct" or "RNAi precursor" refers
to an RNAi-inducing compound such as small interfering RNAs (siRNAs),
hairpin RNAs, and other RNA species which can be cleaved in vivo to form
a siRNA. RNAi precursors herein also include expression vectors (also
referred to as RNAi expression vectors) capable of giving rise to
transcripts which form dsRNAs or hairpin RNAs in cells, and/or
transcripts which can produce siRNAs in vivo.

[0128]A siHybrid molecule is a double-stranded nucleic acid that has a
similar function to siRNA. Instead of a double-stranded RNA molecule, a
siHybrid is comprised of an RNA strand and a DNA strand. Preferably, the
RNA strand is the antisense strand which binds to a target mRNA. The
siHybrid created by the hybridization of the DNA and RNA strands have a
hybridized complementary portion and preferably at least one 3'
overhanging end.

[0129]siNAs for use within the invention can be assembled from two
separate oligonucleotides, where one strand is the sense strand and the
other is the antisense strand, wherein the antisense and sense strands
are self-complementary (i.e., each strand comprises nucleotide sequence
that is complementary to nucleotide sequence in the other strand; such as
where the antisense strand and sense strand form a duplex or double
stranded structure, for example wherein the double stranded region is
about 19 base pairs). The antisense strand may comprise a nucleotide
sequence that is complementary to a nucleotide sequence in a target
nucleic acid molecule or a portion thereof, and the sense strand may
comprise a nucleotide sequence corresponding to the target nucleic acid
sequence or a portion thereof. Alternatively, the siNA can be assembled
from a single oligonucleotide, where the self-complementary sense and
antisense regions of the siNA are linked by means of a nucleic acid-based
or non-nucleic acid-based linker(s).

[0130]In some embodiments, siNAs for intracellular delivery can be a
polynucleotide with a duplex, asymmetric duplex, hairpin or asymmetric
hairpin secondary structure, having self-complementary sense and
antisense regions, wherein the antisense region comprises a nucleotide
sequence that is complementary to a nucleotide sequence in a separate
target nucleic acid molecule or a portion thereof, and the sense region
comprises a nucleotide sequence corresponding to the target nucleic acid
sequence or a portion thereof.

[0132]The antisense region of a siNA molecule can include a
phosphorothioate internucleotide linkage at the 3'-end of said antisense
region. The antisense region can comprise about one to about five
phosphorothioate internucleotide linkages at the 5'-end of said antisense
region. The 3'-terminal nucleotide overhangs of a siNA molecule can
include ribonucleotides or deoxyribonucleotides that are
chemically-modified at a nucleic acid sugar, base, or backbone. The
3'-terminal nucleotide overhangs can include one or more universal base
ribonucleotides. The 3'-terminal nucleotide overhangs can comprise one or
more acyclic nucleotides.

[0133]For example, a chemically-modified siNA can have 1, 2, 3, 4, 5, 6,
7, 8, or more phosphorothioate internucleotide linkages in one strand, or
can have 1 to 8 or more phosphorothioate internucleotide linkages in each
strand. The phosphorothioate internucleotide linkages can be present in
one or both oligonucleotide strands of the siNA duplex, for example in
the sense strand, the antisense strand, or both strands.

[0134]siNA molecules can comprise one or more phosphorothioate
internucleotide linkages at the 3'-end, the 5'-end, or both of the 3'-
and 5'-ends of the sense strand, the antisense strand, or in both
strands. For example, an exemplary siNA molecule can include 1, 2, 3, 4,
5, or more consecutive phosphorothioate internucleotide linkages at the
5'-end of the sense strand, the antisense strand, or both strands.

[0135]In some embodiments, a siNA molecule includes 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, or more pyrimidine phosphorothioate internucleotide linkages in
the sense strand, the antisense strand, or in both strands.

[0136]In some embodiments, a siNA molecule includes 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, or more purine phosphorothioate internucleotide linkages in the
sense strand, the antisense strand, or in both strands.

[0137]A siNA molecule can include a circular nucleic acid molecule,
wherein the siNA is about 38 to about 70, for example, about 38, 40, 45,
50, 55, 60, 65, or 70 nucleotides in length, having about 18 to about 23,
for example, about 18, 19, 20, 21, 22, or 23 base pairs, wherein the
circular oligonucleotide forms a dumbbell-shaped structure having about
19 base pairs and 2 loops.

[0138]A circular siNA molecule can contain two loop motifs, wherein one or
both loop portions of the siNA molecule is biodegradable. For example,
the loop portions of a circular siNA molecule may be transformed in vivo
to generate a double-stranded siNA molecule with 3'-terminal overhangs,
such as 3'-terminal nucleotide overhangs comprising about 2 nucleotides.

[0140]Chemically modified nucleotides can be resistant to nuclease
degradation while at the same time maintaining the capacity to mediate
RNAi.

[0141]The sense strand of a double stranded siNA molecule may have a
terminal cap moiety such as an inverted deoxyabasic moiety, at the
3'-end, 5'-end, or both 3' and 5'-ends of the sense strand.

[0142]Examples of conjugates include conjugates and ligands described in
Vargeese, et al., U.S. application Ser. No. 10/427,160, filed Apr. 30,
2003, incorporated by reference herein in its entirety, including the
drawings.

[0143]In some embodiments of this invention, the conjugate may be
covalently attached to the chemically-modified siNA molecule via a
biodegradable linker. For example, the conjugate molecule may be attached
at the 3'-end of either the sense strand, the antisense strand, or both
strands of the chemically-modified siNA molecule.

[0144]In some embodiments, the conjugate molecule is attached at the
5'-end of either the sense strand, the antisense strand, or both strands
of the chemically-modified siNA molecule. In some embodiments, the
conjugate molecule is attached both the 3'-end and 5'-end of either the
sense strand, the antisense strand, or both strands of the
chemically-modified siNA molecule, or any combination thereof.

[0145]In some embodiments, a conjugate molecule comprises a molecule that
facilitates delivery of a chemically-modified siNA molecule into a
biological system, such as a cell.

[0146]In some embodiments, a conjugate molecule attached to the
chemically-modified siNA molecule is a polyethylene glycol, human serum
albumin, or a ligand for a cellular receptor that can mediate cellular
uptake. Examples of specific conjugate molecules contemplated by the
instant invention that can be attached to chemically-modified siNA
molecules are described in Vargeese, et al., U.S. Patent Publication No.
20030130186 and U.S. Patent Publication No. 20040110296, which are each
hereby incorporated by reference in their entirety.

[0147]A siNA may be contain a nucleotide, non-nucleotide, or mixed
nucleotide/non-nucleotide linker that joins the sense region of the siNA
to the antisense region of the siNA. In some embodiments, a nucleotide
linker can be 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In some
embodiments, the nucleotide linker can be a nucleic acid aptamer. As used
herein, the terms "aptamer" or "nucleic acid aptamer" encompass a nucleic
acid molecule that binds specifically to a target molecule, wherein the
nucleic acid molecule contains a sequence that is recognized by the
target molecule in its natural setting. Alternately, an aptamer can be a
nucleic acid molecule that binds to a target molecule where the target
molecule does not naturally bind to a nucleic acid.

[0150]A "non-nucleotide linker" refers to a group or compound that can be
incorporated into a nucleic acid chain in the place of one or more
nucleotide units, including either sugar and/or phosphate substitutions,
and allows the remaining bases to exhibit their enzymatic activity. The
group or compound can be abasic in that it does not contain a commonly
recognized nucleotide base, such as adenosine, guanine, cytosine, uracil
or thymine, for example at the C1 position of the sugar.

[0152]siNA molecules, which can be chemically-modified, can be synthesized
by: (a) synthesis of two complementary strands of the siNA molecule; and
(b) annealing the two complementary strands together under conditions
suitable to obtain a double-stranded siNA molecule. In some embodiments,
synthesis of the complementary portions of the siNA molecule is by solid
phase oligonucleotide synthesis, or by solid phase tandem oligonucleotide
synthesis.

[0154]An "asymmetric hairpin" as used herein is a linear siNA molecule
comprising an antisense region, a loop portion that can comprise
nucleotides or non-nucleotides, and a sense region that comprises fewer
nucleotides than the antisense region to the extent that the sense region
has enough complementary nucleotides to base pair with the antisense
region and form a duplex with loop.

[0155]An "asymmetric duplex" as used herein is a siNA molecule having two
separate strands comprising a sense region and an antisense region,
wherein the sense region comprises fewer nucleotides than the antisense
region to the extent that the sense region has enough complementary
nucleotides to base pair with the antisense region and form a duplex.

[0156]To "modulate gene expression" as used herein is to upregulate or
down-regulate expression of a target gene, which can include upregulation
or downregulation of mRNA levels present in a cell, or of mRNA
translation, or of synthesis of protein or protein subunits, encoded by
the target gene.

[0157]The terms "inhibit", "down-regulate", or "reduce expression," as
used herein mean that the expression of the gene, or level of RNA
molecules or equivalent RNA molecules encoding one or more proteins or
protein subunits, or level or activity of one or more proteins or protein
subunits encoded by a target gene, is reduced below that observed in the
absence of the nucleic acid molecules (e.g., siNA) of the invention.

[0158]"Gene silencing" as used herein refers to partial or complete
inhibition of gene expression in a cell and may also be referred to as
"gene knockdown." The extent of gene silencing may be determined by
methods known in the art, some of which are summarized in International
Publication No. WO 99/32619.

[0159]As used herein, the terms "ribonucleic acid" and "RNA" refer to a
molecule containing at least one ribonucleotide residue. A ribonucleotide
is a nucleotide with a hydroxyl group at the 2' position of a
beta-D-ribo-furanose moiety. These terms include double-stranded RNA,
single-stranded RNA, isolated RNA such as partially purified RNA,
essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well
as modified and altered RNA that differs from naturally occurring RNA by
the addition, deletion, substitution, modification, and/or alteration of
one or more nucleotides. Alterations of an RNA can include addition of
non-nucleotide material, such as to the end(s) of a siNA or internally,
for example at one or more nucleotides of an RNA.

[0160]Nucleotides in an RNA molecule include non-standard nucleotides,
such as non-naturally occurring nucleotides or chemically synthesized
nucleotides or deoxynucleotides. These altered RNAs can be referred to as
analogs.

[0161]By "highly conserved sequence region" is meant, a nucleotide
sequence of one or more regions in a target gene does not vary
significantly from one generation to the other or from one biological
system to the other.

[0162]By "sense region" is meant a nucleotide sequence of a siNA molecule
having complementarity to an antisense region of the siNA molecule. In
addition, the sense region of a siNA molecule can comprise a nucleic acid
sequence having homology with a target nucleic acid sequence.

[0163]By "antisense region" is meant a nucleotide sequence of a siNA
molecule having complementarity to a target nucleic acid sequence. In
addition, the antisense region of a siNA molecule can include a nucleic
acid sequence having complementarity to a sense region of the siNA
molecule.

[0164]By "target nucleic acid" is meant any nucleic acid sequence whose
expression or activity is to be modulated. A target nucleic acid can be
DNA or RNA.

[0165]By "complementarity" is meant that a nucleic acid can form hydrogen
bond(s) with another nucleic acid sequence either by traditional
Watson-Crick or by other non-traditional modes of binding.

[0166]The term "biodegradable linker" as used herein, refers to a nucleic
acid or non-nucleic acid linker molecule that is designed as a
biodegradable linker to connect one molecule to another molecule, for
example, a biologically active molecule to a siNA molecule or the sense
and antisense strands of a siNA molecule. The biodegradable linker is
designed such that its stability can be modulated for a particular
purpose, such as delivery to a particular tissue or cell type. The
stability of a nucleic acid-based biodegradable linker molecule can be
variously modulated, for example, by combinations of ribonucleotides,
deoxyribonucleotides, and chemically-modified nucleotides, such as
2'-O-methyl, 2'-fluoro, 2'-amino, 2'-O-amino, 2'-C-allyl, 2'-O-allyl, and
other 2'-modified or base modified nucleotides. The biodegradable nucleic
acid linker molecule can be a dimer, trimer, tetramer or longer nucleic
acid molecule, for example, an oligonucleotide of about 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in
length, or can comprise a single nucleotide with a phosphorus-based
linkage, for example, a phosphoramidate or phosphodiester linkage. The
biodegradable nucleic acid linker molecule can also comprise nucleic acid
backbone, nucleic acid sugar, or nucleic acid base modifications.

[0167]In connection with 2'-modified nucleotides as described herein, by
"amino" is meant 2'-NH2 or 2'-O--NH2, which can be modified or
unmodified. Such modified groups are described, for example, in Eckstein,
et al., U.S. Pat. No. 5,672,695 and Matulic-Adamic, et al., U.S. Pat. No.
6,248,878.

[0169]Nucleic acid molecules and peptides can be administered to cells by
a variety of methods known to those of skill in the art, including, but
not restricted to, administration within formulations that comprise the
siNA and peptide alone, or that further comprise one or more additional
components, such as a pharmaceutically acceptable carrier, diluent,
excipient, adjuvant, emulsifier, buffer, stabilizer, preservative, and
the like. In certain embodiments, the siNA and/or the peptide can be
encapsulated in liposomes, administered by iontophoresis, or incorporated
into other vehicles, such as hydrogels, cyclodextrins, biodegradable
nanocapsules, bioadhesive microspheres, or proteinaceous vectors (see
e.g., O'Hare and Normand, International PCT Publication No. WO 00/53722).
Alternatively, a nucleic acid/peptide/vehicle combination can be locally
delivered by direct injection or by use of an infusion pump. Direct
injection of the nucleic acid molecules of the invention, whether
subcutaneous, intramuscular, or intradermal, can take place using
standard needle and syringe methodologies, or by needle-free technologies
such as those described in Conry et al., Clin. Cancer Res. 5:2330-2337,
1999, and Barry, et al., International PCT Publication No: WO 99/31262.

[0170]The compositions of the instant invention can be effectively
employed as pharmaceutical agents. Pharmaceutical agents prevent,
modulate the occurrence or severity of, or treat (alleviate one or more
symptom(s) to a detectable or measurable extent) of a disease state or
other adverse condition in a patient.

[0171]Thus within additional embodiments the invention provides
pharmaceutical compositions and methods featuring the presence or
administration of one or more polynucleic acid(s), typically one or more
siNAs, combined, complexed, or conjugated with a peptide, optionally
formulated with a pharmaceutically-acceptable carrier, such as a diluent,
stabilizer, buffer, and the like.

[0172]The present invention satisfies additional objects and advantages by
providing short interfering nucleic acid (siNA) molecules that modulate
expression of genes associated with a particular disease state or other
adverse condition in a subject. Typically, the siNA will target a gene
that is expressed at an elevated level as a causal or contributing factor
associated with the subject disease state or adverse condition. In this
context, the siNA will effectively downregulate expression of the gene to
levels that prevent, alleviate, or reduce the severity or recurrence of
one or more associated disease symptoms. Alternatively, for various
distinct disease models where expression of the target gene is not
necessarily elevated as a consequence or sequel of disease or other
adverse condition, down regulation of the target gene will nonetheless
result in a therapeutic result by lowering gene expression (i.e., to
reduce levels of a selected mRNA and/or protein product of the target
gene). Alternatively, siNAs of the invention may be targeted to lower
expression of one gene, which can result in upregulation of a
"downstream" gene whose expression is negatively regulated by a product
or activity of the target gene.

[0173]This siNAs of the present invention may be administered in any form,
for example transdermally or by local injection. Comparable methods and
compositions are provided that target expression of one or more different
genes associated with a selected disease condition in animal subjects,
including any of a large number of genes whose expression is known to be
aberrantly increased as a causal or contributing factor associated with
the selected disease condition.

[0174]Negatively charged polynucleotides of the invention (e.g., RNA or
DNA) can be administered to a patient by any standard means, with or
without stabilizers, buffers, and the like, to form a pharmaceutical
composition. When it is desired to use a liposome delivery mechanism,
standard protocols for formation of liposomes can be followed. The
compositions of the present invention may also be formulated and used as
tablets, capsules or elixirs for oral administration, suppositories for
rectal administration, sterile solutions, suspensions for injectable
administration, and the other compositions known in the art.

[0175]The present invention also includes pharmaceutically acceptable
formulations of the compositions described herein. These formulations
include salts of the above compounds, e.g., acid addition salts, for
example, salts of hydrochloric, hydrobromic, acetic acid, and benzene
sulfonic acid.

[0176]The siNAs can also be administered in the form of suppositories,
e.g., for rectal administration of the drug. These compositions can be
prepared by mixing the drug with a suitable non-irritating excipient that
is solid at ordinary temperatures but liquid at the rectal temperature
and will therefore melt in the rectum to release the drug. Such materials
include cocoa butter and polyethylene glycols.

[0177]Nucleic acid molecules can be administered to cells by a variety of
methods known to those of skill in the art, including, but not restricted
to, encapsulation in liposomes, by iontophoresis, or by incorporation
into other vehicles, such as biodegradable polymers, hydrogels,
cyclodextrins (see for example, Gonzalez, et al., Bioconjugate Chem.
10:1068-1074, 1999; Wang, et al., International PCT Publication Nos. WO
03/47518 and WO 03/46185), poly(lactic-co-glycolic acid) (PLGA) and PLCA
microspheres (see for example, U.S. Pat. No. 6,447,796 and U.S. Patent
Application Publication No. US 2002130430), biodegradable nanocapsules,
and bioadhesive microspheres, or by proteinaceous vectors (O'Hare and
Normand, International PCT Publication No. WO 00/53722). Alternatively,
the nucleic acid/vehicle combination is locally delivered by direct
injection or by use of an infusion pump. Direct injection of the nucleic
acid molecules of the invention, whether subcutaneous, intramuscular, or
intradermal, can take place using standard needle and syringe
methodologies, or by needle-free technologies such as those described in
Conry, et al., Clin. Cancer Res. 5:2330-2337, 1999, and Barry, et al.,
International PCT Publication No. WO 99/31262. The molecules of the
instant invention can be used as pharmaceutical agents. Pharmaceutical
agents prevent, modulate the occurrence, or treat (alleviate a symptom to
some extent, preferably all of the symptoms) of a disease state in a
subject.

[0178]Any one or combination of the cationic peptides of the present
invention may be selected or combined to yield effective polynucleotide
delivery-enhancing polypeptide reagents to induce or facilitate
intracellular delivery of siNAs within the methods and compositions of
the invention.

Pharmaceutical Composition

[0179]The present invention also includes pharmaceutically acceptable
formulations or compositions of the compounds described herein. These
formulations include organic and inorganic salts of the above compounds,
e.g., acid addition salts, for example, salts of hydrochloric,
hydrobromic, acetic acid, and benzene sulfonic acid.

[0180]Aqueous suspensions contain the active materials in admixture with
excipients suitable for the manufacture of aqueous suspensions. Such
excipients are suspending agents, for example sodium
carboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose,
sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;
dispersing or wetting agents can be a naturally-occurring phosphatide,
for example, lecithin, or condensation products of an alkylene oxide with
fatty acids, for example polyoxyethylene stearate, or condensation
products of ethylene oxide with long chain aliphatic alcohols, for
example heptadecaethyleneoxycetanol, or condensation products of ethylene
oxide with partial esters derived from fatty acids and a hexitol such as
polyoxyethylene sorbitol monooleate, or condensation products of ethylene
oxide with partial esters derived from fatty acids and hexitol
anhydrides, for example polyethylene sorbitan monooleate. The aqueous
suspensions can also contain one or more preservatives, for example
ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or
more flavoring agents, and one or more sweetening agents, such as sucrose
or saccharin.

[0181]Oily suspensions can be formulated by suspending the active
ingredients in a vegetable oil, for example arachis oil, olive oil,
sesame oil or coconut oil, or in a mineral oil such as liquid paraffin.
The oily suspensions can contain a thickening agent, for example beeswax,
hard paraffin or cetyl alcohol. Sweetening agents and flavoring agents
can be added to provide palatable oral preparations. These compositions
can be preserved by the addition of an anti-oxidant such as ascorbic
acid.

[0182]Dispersible powders and granules suitable for preparation of an
aqueous suspension by the addition of water provide the active ingredient
in admixture with a dispersing or wetting agent, suspending agent and one
or more preservatives. Additional excipients, for example sweetening,
flavoring and coloring agents, can also be present.

[0183]Pharmaceutical compositions of this invention can also be in the
form of oil-in-water emulsions. The oily phase can be a vegetable oil, or
a mineral oil, or mixtures thereof. Suitable emulsifying agents can be
naturally-occurring gums, for example gum acacia or gum tragacanth,
naturally-occurring phosphatides, for example soy bean, lecithin, and
esters or partial esters derived from fatty acids and hexitol,
anhydrides, for example sorbitan monooleate, and condensation products of
the said partial esters with ethylene oxide, for example polyoxyethylene
sorbitan monooleate. The emulsions can also contain sweetening and
flavoring agents.

[0184]The pharmaceutical compositions can be in the form of a sterile
injectable aqueous or oleaginous suspension. This suspension can be
formulated using a suitable dispersing or wetting agent, and/or a
suspending agent. A sterile injectable preparation can also be a sterile
injectable solution or suspension in a non-toxic parentally acceptable
diluent or solvent, for example as a solution in 1,3-butanediol.

[0185]Among the acceptable carriers, vehicles and solvents for a
pharmaceutical composition that can be employed are water, Ringer's
solution, and isotonic sodium chloride solution. In addition, sterile,
fixed oils are conventionally employed as a carrier, vehicle, solvent, or
suspending medium. For this purpose, any bland fixed oil can be employed
including synthetic mono- or diglycerides. In addition, fatty acids such
as oleic acid find use in the preparation of injectables.

[0186]All publications, references, patents, and patent applications cited
herein are each hereby specifically incorporated by reference in their
entirety.

[0187]While this invention has been described in relation to certain
embodiments; and many details have been set forth for purposes of
illustration, it will be apparent to those skilled in the art that this
invention includes additional embodiments, and that some of the details
described herein may be varied considerably without departing from this
invention. This invention includes such additional embodiments,
modifications and equivalents.

[0188]The use herein of the terms "a," "an," "the," and similar terms in
describing the invention, and in the claims, are to be construed to
include both the singular and the plural. The terms "comprising,"
"having," "including," and "containing" are to be construed as open-ended
terms which mean, for example, "including, but not limited to."
Recitation of a range of values herein is intended to refer individually
to each separate value falling within the range as if it were
individually recited herein, whether or not some of the values within the
range are expressly recited. Specific values employed herein will be
understood as exemplary and not to limit the scope of the invention.

[0189]The examples given herein, and the exemplary language used herein
are solely for the purpose of illustration, and are not intended to limit
the scope of the invention.

EXAMPLES

Preparation Example 1

[0190]PN0826:siRNA compounds in water. A compound was prepared by: Adding
82.12 μl of RNase free water to a centrifuge tube, and then 10 μl
of G1498 (1 mg/ml, in RNase free water). The solution was vortexed to
mix. Finally, 7.88 μl of PN0826 was added (1 mg/ml, in RNase free
water) and vortexed to mix.

[0194]G1498, PN0183, water for dilution and peptide first. A compound was
prepared by: in a centrifuge tube, 85.83 μl of 10 mM Hepes/5% dextrose
buffer (pH5.0) was added first, then 4.17 μl of peptide PN0183 (5
mg/ml, in RNase free water), and vortexed to mix. Finally, 10 μl G1498
(1 mg/ml, in RNase free water) was added to the solution and vortexed
again to mix.

[0216]The relative binding of various peptides to siRNA via a rapid screen
was assessed by indirect measurement of the displacement of SYBR-gold
nucleic acid binding dye. A buffered mixture of siRNA, peptide and
SYBR-gold was prepared in the measurement plate in duplicate such that
the peptide and SYBR-gold dye underwent simultaneous competitive binding
of the siRNA. The concentration of siRNA was fixed at 10 μg/mL and was
combined with a titration of each peptide ranging in a concentration that
corresponded to a peptide:siRNA charge ratio between 0.05 and 10. Since
SYBR-gold dye only fluoresces when bound to siRNA, peptide binding to the
siRNA inhibits binding of the dye and consequently reduces the
fluorescence. Therefore, the amount of fluorescence correlated inversely
to the binding of the peptide to the siRNA. Both Kd and Bmax values
were calculated. A greater Kd value indicated greater binding affinity
between the peptide and the siRNA.

[0217]SYBR-gold nucleic acid binding dye stock, a 10,000×
concentrate, was supplied by Invitrogen (Carlsbad, Calif.) and stored at
-20° C. The concentrate was allowed to equilibrate to room
temperature before diluting 1 to 100 in Hyclone nuclease free water. This
was diluted 1 to 10 in the experimental plate for a final concentrate of
10× for the assay. This was the optimal dilution to achieve linear
binding to siRNA duplex at a concentration range of up to 50 μg/mL
concentration. The values used to generate the standard curve
demonstrating linear binding of SYBR-gold to G1498 siRNA are shown in
Table 4.

[0218]Samples were mixed directly in the 384 well analysis plate. First, 5
μL SYBR-gold dye was pipetted into each well with a multichannel
pipet, touching the tip to the bottom of the well to draw out the
solution completely. Second, 22.5 μL, of 2× peptide solution was
added with a single channel pipet. Finally, 22.5 μL of 2× siRNA
was added with a multichannel pipet. The plate was covered immediately
with foil and tapped gently to mix and draw down any droplets on the side
of the well.

[0219]Fluorescence was measured using the SpectraMax fluorescent plate
reader from Molecular Devices (Sunnyvale, Calif.). Plate settings
included shaking before reading, one read per well, with excitation
wavelength of 495 nm and emission wavelength of 537 nm. The plate was
read within 30 minutes of the addition of the siRNA.

Scatchard Plot for Peptide Binding

[0220]A Scatchard Plot is a plot of peptide binding
([peptide]bound/[peptide]free) vs. [peptide]bound. The slope of the
linear regression of this plot is -1/Kd and Bmax is the y-intercept.
Since the concentration of free and bound peptide cannot be measured
directly, indirect measurement of siRNA was used for the calculation.
Free siRNA was determined from measured fluorescence using the standard
curve. Bound siRNA was determined from the standard curve by mass balance
from the known initial siRNA concentration (10 μg/mL).

[0221]Bound peptide was calculated from bound siRNA by assuming the
(siRNA:Peptide) bound molar ratio was equal to the (siRNA:Peptide) charge
ratio for single molecule pairing. From this calculated bound peptide
amount, the free peptide was calculated by mass balance.

Particle Size and Zeta Potential

[0222]Particle size and zeta potential were determined with a Malvern
Zetasizer Nano ZS (Malvern, Worcestershire, UK) using a DTS1060C clear
disposable zeta cell at 25°. The dispersant for particle size was
PBS, 1.0200 CP viscosity, or water, 0.8872 CP viscosity. The dispersant
for zeta potential was water 0.8872 CP viscosity. The dispersant
viscosity was used as the sample viscosity. When both the zeta potential
and the particle size were measured, the clear disposable zeta cell was
used. When only the particle size was measured, then a low volume
disposable sizing cuvette was used.

[0223]Diameters of particles of a condensate compound of siRNA G1498 and
peptide PN183 at various concentrations of G1498 and various N/P ratios
are shown in FIG. 1. For each group of three bars at a particular N/P,
the concentration of G1498 for the leftmost bar was 100 ug/ml, for the
middle bar was 50 ug/ml, and for the rightmost bar was 10 ug/ml. At N/P
of 0.2 and 0.5, the particles were very small when the concentration of
G1498 was 10 ug/ml, thus the bar does not appear.

[0224]At N/P ratios below about 1.4, the particle size was below about 200
nm for all concentrations of the siRNA. At N/P ratios at or above about
1.4, condensate particle size remained below about 200 nm for all
concentrations of RNA except the highest (100 ug/ml).

[0225]Diameters of particles of condensate compounds of siRNA G1498 and
peptide PN183 obtained at various times after mixing and at various
nitrogen to phosphorous ratios (N/P) are shown in FIGS. 2-5. In each of
FIGS. 2-5, for each group of two bars at a particular N/P ratio, the left
bar was with vortexing, while the right bar was without vortexing.

[0226]The particles sizes in FIG. 2 were obtained immediately after
mixing, while those of FIGS. 3, 4, and 5 were obtained 30 minutes, 60
minutes, and 24 hours after mixing, respectively.

Example 4

Effect of pH on Condensate Particle Size

[0227]Diameters of particles of condensate compounds of siRNA G1498 and
peptide PN183 obtained at an N/P ratio of 1.4 and at a concentration of
G1498 of 100 ug/ml for various values of pH are shown in FIG. 6.

[0229]The intensity is the measure of back scattered photons (backscatter
mode). The particle size is calculated size using an algorithm for the
diffusion autocorrelation.

Example 5

Effect of Salt Concentration on Condensate Particle Size

[0230]Diameters of particles of condensate compounds of siRNA G1498 and
peptide PN183 obtained at various concentrations of sodium chloride are
shown in FIG. 7.

[0231]At sodium chloride concentrations up to about 0.5, particle size
increases from about 100 nm to about 275 nm. At sodium chloride
concentrations greater than about 0.5, condensate particle size
fluctuates.

Example 6

Effect of Order of Addition of RNA and Peptide on Condensate Particle Size

[0232]Diameters of particles of condensate compounds of siRNA G1498 with
peptide PN183 at various N/P ratios and order of mixing are shown in FIG.
8. At N/P ratios at or below about 0.5, particle size was not much
affected by the order of addition. At N/P ratios above about 0.5,
particle size was generally smaller when the siRNA was introduced to the
solution first, and the peptide was added to the siRNA solution.

Example 7

Morphology of Condensate Particles

[0233]The morphology of the particles of a peptide-RNA condensate compound
was determined by transmission electron microscopy (TEM) imaging. The
following protocol was used:

[0236]Using the protocol above with no glow discharge, a TEM of particles
of a condensate compound of siRNA G1498 (concentration 100 ug/ml) with
peptide PN183 (N/P=1.4) is shown in FIG. 9. This image shows particles of
uniform size and spherical morphology. The particle size was below 100
nm, typically about 50-60 nm.

[0237]Using the protocol above with glow discharge, a TEM of particles of
a condensate compound of siRNA G1498 (concentration 100 ug/ml) with
peptide PN183 (N/P=1.4) is shown in FIG. 10. This image shows particles
of uniform size and spherical morphology. The particle size is below 100
nm, typically about 30-60 nm.

In vivo Knockdown Assay of TFN-α in LPS-Stimulated Mouse Lung Using
Peptide-RNA Condensates

[0240]SiRNA knockdown activity was determined by transfecting cells with a
peptide-siRNA condensate compound. A random siRNA sequence was used as a
negative control.

[0241]FIG. 11 shows the results of a knockdown assay of LPS-induced
TFN-α expression (pg/ml) in a mouse model by intranasal
administration of a composition including a condensate compound of siRNA
Inm-4 and peptides PN183 and PN939.

[0242]In FIG. 11, buffer control is the leftmost bar, followed by data for
condensate Inm-4/PN183/PN939, and followed on the right by data for
compound Inm-4/PN183/PN939 crosslinked with glutaraldehyde (G). Placebo
does not contain the siRNA, and Qneg contains a non-active-siRNA.

[0262]FIG. 12 shows the results of a knockdown in vitro assay of lac-z
expression in rat gliosarcoma fibroblast cells 9L/LacZ for condensate
compounds of the lac-z siRNA with peptide PN183 and various second
peptides.

[0263]In FIG. 12, comparative data using HiPerFect® (Qiagen; Valencia,
Calif.) is the leftmost bar, followed by data for various compounds of
this invention. The N/P ratio for PN183 was 0.75, while the N/P ratio for
the second peptide was 0.3. The data for FIG. 12 are given in Table 10.

[0281]Materials and methods for the assay were as follows: [0282]Cells:
9L/LacZ. [0283]Dose: 100 nM; based on 100 ul total transfection volume.
[0284]Volume: 25 uL formulation volume. [0285]Replicates: n=3.
[0286]Total Groups: 20. [0287]Controls: Qneg w/ Alexis 546. [0288]siRNA:
LacZ. [0289]Transfection: Each formulation had 125 ul which was enough
for 5 wells. Each well (n=3) received 25 ul. [0290]Peptide Preparation
Peptide was diluted into appropriate concentration using OPTI-MEM medium.
[0291]Excipient Preparation All the excipients were 0.2 um filtered to be
sterile. [0292]Formulation Preparation: [0293](A) For formulations
without PN0183, added delivery vehicle first and then siRNA, pipetted to
mix. [0294](B) For formulations with PN0183, made siRNA and PN0183
complex first. In 96 well plate, added delivery vehicles first, and then
siRNA/PN0183 complex, pipetted to mix. [0295](C) For formulations with
crosslinking, make the siRNA/PN0183 complex first, then either dialysis
(at 4°, overnight), or without dialysis. Then next morning, added
other delivery vehicles first and siRNA/PN0183 complex, and then pipetted
to mix.

[0298]The exemplary polynucleotide delivery-enhancing polypeptide PN73 was
derived from the amino acid sequence of the human histone 2B (H2B)
protein, which is shown below. The underlined residues 13 through 48
found within H2B protein identify the fragment used to derive PN73. It
may also be represented by H2B amino acids 12 through 48. The primary
structure of PN73 is also shown below.

[0299]Table 18 shows the structure of some mutant polynucleotide
delivery-enhancing polypeptides made by residue substitutions and
deletions of the exemplary polynucleotide delivery-enhancing polypeptide
PN73.

[0301]PN360 shares its N-terminus with PN73 but lacks PN73's C-terminus
while PN361 shares its C-terminus with PN73 but lacks PN73's N-terminus.
PN766 represents the 15 C-terminal amino acids of PN73. PN73, PN360,
PN361 and PN766 are not tagged with a C-terminal FITC
(fluorescein-5-isothiocyanate) (i.e., -GK[EPSILON]G-amide). Table 19
further shows the 11 truncated forms of PN73 that were created by
sequentially deleting 3 residues at a time, except PN768, from the
N-terminus of the peptide. All these peptides were tagged with a
C-terminus FITC (fluorescein-5-isothiocyanate) label (i.e.,
-GK[EPSILON]G-amide) so that cells containing the peptide could be
detected by fluorescent microscopy and/or sorted by flow cytometry. PN766
and PN708 have the same amino acid sequence but differ in that PN708 has
the C-terminus FITC tag.

Example 13

In Vitro Methods and Procedures for siRNA Cell-Uptake and Target Gene
Knockdown

[0302]The present example illustrates the methods and procedures used to
assess the efficacy of the exemplary polynucleotide delivery-enhancing
polypeptides listed in Table 18 and Table 19 of Example 12 to enhance
siRNA cell-uptake and siRNA mediated target gene knockdown activities.
Cell viability was also assessed. The cell culture conditions and
protocols for each assay are explained below in detail.

Cell Cultures

[0303]Primary Human Monocytes: Fresh human blood samples from healthy
donors were purchased from Golden West Biologicals. For isolation of
monocytes, blood samples were diluted with PBS at a 1:1 ratio immediately
after receiving. Peripheral blood mononuclear cells (PBMC) were first
isolated by Ficoll (Amersham) gradient from whole blood. Monocytes were
further purified from PBMCs using the Miltenyi CD14 positive selection
kit and supplied protocol (MILTENYI BIOTEC). To asses the purity of the
monocyte preparation, cells were incubated with an anti-CD14 antibody (BD
Biosciences) and then sorted by flow cytometry. The purity of the
monocyte preparation was greater than 95%.

[0304]Activation of human monocytes was performed by adding 0.1-1.0 ng/ml
of Liposaccharides, LPS (Sigma, St Louis, Mo.) to the cell culture to
stimulate tumor necrosis factor-±(TNF-±) production. Cells were
harvested 3 hours after incubation with LPS and mRNA levels were
determined by Quantigene assay (Genospectra, Fremont, Calif.) according
to the manufacturer's instructions.

[0305]Mouse Tail Fibroblast Cells: Mouse tail fibroblast (MTF) cells were
derived from the tails of C57BL/6J mice. Tails were removed, immersed in
70% ethanol and then cut into small sections with a razor blade. The
sections were washed three times with PBS and then incubated in a shaker
at 37° C. with 0.5 mg/mL collagenase, 100 units/mL penicillin and
100 μg/mL streptomycin to disrupt tissue. Tail sections were then
cultured in complete media (Dulbecco's Modified Essential Medium with 20%
FBS, 1 mM sodium pyruvate, nonessential amino acids and 100 units/mL
penicillin and 100 μg/mL streptomycin) until cells were established.
Cells were cultured at 37° C., 5% CO2 in complete media as
outlined above.

Cell Viability (MTT Assay)

[0306]Cell viability was assessed using the MTT assay (MTT-100, MatTek
kit). This kit measures the uptake and transformation of tetrazolium salt
to formazan dye. Thawed and diluted MTT concentrate was prepared 1 hour
prior to the end of the dosing period with the lipid by mixing 2 mL of
MTT concentrate with 8 mL of MTT diluent. Each cell culture insert was
washed twice with PBS containing Ca+2 and Mg+2 and then
transferred to a new 96-well transport plate containing 100 μL of the
mixed MTT solution per well. This 96-well transport plate was then
incubated for 3 hours at 37° C. and 5% CO2. After the 3 hour
incubation, the MTT solution was removed and the cultures transferred to
a second 96-well feeder tray containing 250 μL MTT extractant solution
per well. An additional 150 μL of MTT extractant solution was added to
the surface of each culture well and the samples sat at room temperature
in the dark for a minimum of 2 hours and maximum of 24 hours. The insert
membrane was then pierced with a pipet tip and the solutions in the upper
and lower wells were allowed to mix. Two hundred microliters of the mixed
extracted solution along with extracted blanks (negative control) was
transferred to a 96-well plate for measurement with a microplate reader.
The optical density (OD) of the samples was measured at 570 nm with the
background subtraction at 650 nm on a plate reader. Cell viability was
expressed as a percentage and calculated by dividing the OD readings for
treated inserts by the OD readings for the PBS treated inserts and
multiplying by 100. For the purposes of this assay, it was assumed that
PBS had no effect on cell viability and therefore represented 100% cell
viability.

siRNA Preparation

[0307]Synthesis of oligonucleotides was carried out using the standard
2-cyanoethyl phosphoramidite method (1) on long chain alkylamine
controlled pore glass derivatized with
5'-O-Dimethyltrityl-2'-O-t-butyldimethylsilyl-3'-O-succinyl
ribonucleoside of choice or 5'-O-Dimethyltrityl-2'-deoxy-3'-O-succinyl
thymidine support where applicable. All oligonucleotides were synthesized
at either the 0.2 or 1-μmol scale using an ABI 3400 DNA/RNA
synthesizer (Applied Biosystems, Foster City, Calif.), cleaved from the
solid support using concentrated NH4OH, and deprotected using a 3:1
mixture of NH4OH:ethanol at 55° C. The deprotection of
2'-TBDMS protecting groups was achieved by incubating the
base-deprotected RNA with a solution (600 μL per μmol) of
N-methylpyrrolidinone/triethylamine/triethylamine trihydrofluoride
(NMP/TEA/3HF; 6:3:4 by volume) at 65° C. for 2.5 hours. The
corresponding building blocks,
5'-dimethoxytrityl-N-(tac)-2'-O-(t-butyldimethylsilyl)-3'-[(2-cyanoethyl)-
-(N,N-diisopropyl)]-phosphoramidites of A, U, C and G (Proligo, Boulder,
Colo.) as well the modified phosphoramidites,
5'-DMTr-5-methyl-U-TOM-CE-Phosphoramidite, 5'-DMTr-2'-OMe-Ac-C-CE
Phosphoramidite, 5'-DMTr-2'-OMe-G-CE Phosphoramidite, 5'-DMTr-2'-OMe-U-CE
Phosphoramidite, 5'-DMTr-2'-OMe-A-CE Phosphoramidite (Glen Research,
Sterling Va.) were purchased directly from suppliers.
Triethylamine-trihydrofluride, N-methylpyrrolidinone and concentrated
ammonium hydroxide was purchased from Aldrich (Milwaukee, Wis.). All HPLC
analysis and purifications were performed on a Waters 2690 HPLC system
with Xterra® C18 columns. All other reagents were purchased from Glen
Research Inc. Oligonucleotides were purified to greater than 97% purity
as determined by RP-HPLC. siRNAs for mouse injection were purchased from
Qiagen (Valencia, Calif.) as in-vivo grade, which were HPLC purified
after annealing. The amount of single stranded siRNA was determined
spectrophotometrically based on a calculated extinction coefficient of
35.0 μg/OD for the sodium salt form at λ=260 nm. When the two
strands are annealed, approximately 10% hypochromicity was observed;
therefore the extinction coefficient was lowered by 10% for the
quantitation of the double stranded forms. Endotoxin levels of siRNAs
were typically equal to or less than 0.0024 EU/mg.

Peptide Synthesis

[0308]Peptides were synthesized by solid-phase Fmoc chemistry on
CLEAR-amide resin using a Rainin Symphony synthesizer. Coupling steps
were performed using 5 equivalents of HCTU and Fmoc amino acid with an
excess of N-methylmorpholine for 40 minutes. Fmoc removal was
accomplished by treating the peptide resin with 20% piperidine in DMF for
two 10 minutes cycles. Upon completion of the entire peptide, the Fmoc
group was removed with piperidine and washed extensively with DMF.
Maleimido modified peptides were prepared by coupling 3.0 equivalents of
3-maleimidopropionic acid and HCTU in the presence of 6 equivalents of
N-methylmorpholine to the N-terminus of the peptide resin. The extent of
coupling was monitored by the Kaiser test. The peptides were cleaved from
the resin by the addition of 10 mL of TFA containing 2.5% water and 2.5
triisopropyl silane followed by gentle agitation at room temperature for
2 h. The resulting crude peptide was collected by trituration with ether
followed by filtration. The crude product was dissolved in Millipore
water and lyophilized to dryness. The crude peptide was taken up in 15 mL
of water containing 0.05% TFA and 3 mL acetic acid and loaded onto a
Zorbax Rx-C8 reversed-phase (22 mm ID×250 mm, 5 μm particle
size) through a 5 mL injection loop at a flow rate of 5 mL/min. The
purification was accomplished by running a linear AB gradient of 0.1%
B/min where solvent A is 0.05% TFA in water and solvent B is 0.05% TFA in
acetonitrile. The purified peptides were analyzed by HPLC and ESMS.

Flow Cytometry

[0309]Fluorescence activated cell sorting (FACS) analysis were performed
using Beckman Coulter FC500 cell analyzer (Fullerton, Calif.). The
instrument was adjusted according to the fluorescence probes used (FAM or
Cy5 for siRNA and FITC and PE for CD14). Propidium iodide (Fluka, St
Louis) and AnnexinV (R&D systems, Minneapolis) were used as indicators
for cell viability and cytotoxicity. A brief step-by-step protocol is
detailed below. [0310](a) After exposure to the complex of
siRNA/peptide, cells were incubated for at least 3 hours. [0311](b) Wash
cells with 200 μl PBS. [0312](c) Detach cells with 15 μl TE,
incubate at 370C. [0313](d) Re-suspend cells in five wells with 30 μl
FACS solution (PBS with 0.5% BSA, and 0.1% sodium Azide). [0314](e)
Combine all five wells into a tube. [0315](f) Add PI (Propidium iodide) 5
μl into each tube. [0316](g) Analyze the cells with fluorescence
activated cell sorting (FCAS) according to manufacturer's instructions.

[0317]For siRNA uptake analysis, cells were washed with PBS, treated with
trypsin (attached cells only), and then analyzed by flow cytometry.
Uptake of the siRNA designated BA, described above, was also measured by
intensity of Cy5 or FITC fluorescence in the cells and cellular viability
assessed by addition of propidium iodide or AnnexinV-PE. In order to
differentiate the cellular uptake from the membrane insertion of
fluorescence labeled siRNA, trypan blue was used to quench the
fluorescence on the cell membrane surface.

[0318]The efficacy of the full-length and truncated forms of polypeptide
PN73 to enter cells was tested in vitro by a cell-uptake assay with
primary mouse tail fibroblast (MTF) cells. The number of cells in culture
that receive the FITC-labeled peptide was measured by flow cytometry. The
percentage peptide cell-uptake was expressed relative to the total number
of cells present in the culture. In addition, the Mean Fluorescence
Intensity (MFI) was used to evaluate the quantity of FITC-labeled peptide
found within cells. MFI directly correlates with the amount of
FITC-labeled peptide within the cell: higher relative MFI value
correlates with a greater amount of intracellular FITC-labeled peptides.
Peptides were evaluated at 0.63 μM, 2.5 μM and 10 μM
concentrations; PN768 was tested at 2 μM, 10 μM and 50 μM.

[0319]Full-length and truncated forms of the exemplary polynucleotide
delivery-enhancing polypeptide PN73, were exposed to cells the day before
transfection. FITC-tagged peptides were diluted in Opti-MEM® media
(Invitrogen) for about 5 minutes at room temperature and then added to
cells. Cells were transfected for 3 hours at and washed with PBS, treated
with trypsin, and then analyzed by flow cytometry. Cell viability was
determined as above. Cellular uptake was distinguished from the membrane
insertion using trypan blue to quench any fluorescence on the cell
membrane surface.

[0320]For the cell-uptake assay, the full-length FITC-labeled PN73 peptide
(PN690) achieved nearly 100% cell uptake at all tested concentrations (10
μM results shown in Table 20 column entitled "% Peptide Cell-Uptake").
The remaining truncated forms of PN73, at 10 μM concentration except
for PN768 which required 50 μM, achieved a percent cell uptake (values
in parentheses) comparable to that of PN690 indicating that the
N-terminal residues of PN73 are not required for the peptide's ability to
enter cells. The five C-terminal residues of PN73, identified as PN768,
are sufficient for peptide cell-uptake. The truncated forms of PN73 at
0.63 μM showed a decrease in cell uptake activity proportionate to the
length of the peptide. In other words, the general observation of the
peptides tested at a 0.63 μM concentration is that, as the PN73
peptide's length decreased, its cell uptake activity decreased thus
indicating peptide cell-uptake activity is dose dependent.

[0322]Table 20 shows that deleting part of the N-terminus of PN73 (see
PN361) reduced siRNA cell-uptake activity by 50%; and removal of
C-terminal residues (see PN360) reduced siRNA cell-uptake activity. These
data show that the C-terminal domain of the exemplary polynucleotide
deliver-enhancing polypeptide PN73 contributes to nucleotide cell-uptake
activity of the peptide.

[0323]The effective knockdown of target gene expression by
siRNA/polynucleotide delivery-enhancing polypeptide complexes of the
invention was demonstrated. Specifically, the ability of siRNA/peptide
complexes to modulate expression of the human tumor necrosis
factor-α (hTNF-α) gene was assessed. The significance of
targeting the hTNF-α gene is that it is implicated in mediating the
occurrence or progression of rheumatoid arthritis (RA) when
over-expressed in human and other mammalian subjects.

[0324]Human monocytes were used as a model system to determine the effect
of siRNA/peptide complexes on hTNF-α gene expression. Qneg
represents a random siRNA sequence and functioned as the negative
control. The observed Qneg knockdown activity is normalized to 100% (100%
gene expression levels) and the knockdown activity of each of the
following siRNAs A19S21, 21/21 and LC20 was presented as a relative
percentage of the negative control. A19S21, 21/21 and LC20 are siRNAs
that target hTNF-α mRNA. The exemplary polynucleotide
delivery-enhancing polypeptides PN643 (full-length PN73 minus a
C-terminal label), PN690 (full-length PN73 with a C-terminal FITC-label)
and the truncated forms of PN73 from the deletion series, PN660, PN735,
PN654 and PN708 were complexed with the above listed siRNAs to determine
their effect on each siRNA's ability to reduce hTNF-α gene
expression levels in human monocytes. The knockdown activity for the full
length and truncated forms of the exemplary polynucleotide
delivery-enhancing polypeptide PN73 are summarized above in Table 20. A
"+" in the "KD" column indicates that the peptide/siRNA complex had
knockdown activity of 80% of the Qneg negative control siRNA (20%
reduction in mRNA levels compared to the Qneg negative control). A "+/-"
indicates that the peptide/siRNA complex had a knockdown activity of
approximately 90% of the Qneg negative control siRNA (10% reduction in
mRNA levels compared to the Qneg negative control). Finally, a "-"
indicates that the peptide/siRNA complex had no significant knockdown
activity compared to the Qneg negative control.

[0325]Healthy human blood was purchased from Golden West Biologicals (CA),
the peripheral blood mononuclear cells (PBMC) were purified from the
blood using Ficoll-Pague plus (Amersham) gradient. Human monocytes were
then purified from the PBMCs fraction using magnetic microbeads from
Miltenyi Biotech. Isolated human monocytes were resuspended in IMDM
supplemented with 4 mM glutamine, 10% FBS, 1× non-essential amino
acid and 1× pen-strep, and stored at 4C until use.

[0326]In a 96 well flat bottom plate, human monocytes were seeded at
100K/well/100 μl in OptiMEM medium (Invitrogen). Exemplary
polynucleotide delivery-enhancing polypeptides were mixed with 20 nM
siRNA at a molar ratio of 1 to 5 in OptiMEM medium at room temperature
for 5 minutes. At the end of incubation, FBS was added to the mixture
(final 3%), and 50 μl of the mixture was added to the cells. The cells
were incubated at 37° C. for 3 hours. After incubation, the cells
were transferred to V-bottom plate and pelleted at 1500 rpm for 5 min.
The cells were resuspended in growth medium (IMDM with glutamine,
non-essential amino acid, and pen-strep). After an overnight incubation,
the monocytes were stimulated by application of LPS (Sigma) at 1 ng/ml
for 3 hours to increase expression of TNF-α expression levels.
After induction by LPS, cells were collected as above for mRNA
quantification, and supernatant was saved for protein quantification if
desired.

[0327]For mRNA measurement, branch DNA technology from Genospectra (CA)
was used according to manufacturer's specification. To quantitate mRNA
level in the cells, both house keeping gene (cypB) and target gene
(TNF-α) mRNA were measured, and the reading for TNF-α was
normalized with cypB to obtain relative luminescence unit.

[0328]In general, PN643 (full-length non-FITC-labeled PN73) and PN690
(full-length FITC-labeled PN73) had equivalent siRNA knockdown activities
for all siRNAs tested as indicated by "+" in the "KD" column (results
shown in Table 20). Additionally, PN660 had siRNA knockdown activities
for all siRNAs tested that were comparable to PN643 and PN690 indicating
that the removal of the 9 most N-terminal residues of the PN73 peptide
did not affect siRNAs mediated knockdown activity of the targeted
TNF-α mRNA. PN654 showed moderate knockdown activity for both the
A19S21 and 21/21 siRNAs but not for the LC20 siRNA (knockdown activity is
shown by "±" in knockdown activity column). However, the siRNAs
complexed with either PN708 or PN735 resulted in no observable knockdown
activity for any of the siRNAs.

Example 15

Polynucleotide Delivery-Enhancing Polypeptide PN708

[0329]As described above, the cell-uptake assay determines the number of
cells that receive Cy5-conjugated siRNA when complexed with a peptide.
siRNA cell-uptake was assessed by flow cytometry (refer to Example 2 for
details). Uptake was expressed as a percentage calculated by dividing the
number of cells containing Cy5-conjugated siRNA by the total number of
transfected and untransfected cells in culture. Mean Fluorescence
Intensity (MFI) was measured by flow cytometry and determined the amount
of Cy5-conjugated siRNA found within cells. The MFI value directly
correlates with the amount of Cy5-conjugated siRNA within the cell, thus,
a higher MFI value indicates a greater number of Cy5-conjugated siRNA
within the cells.

[0330]In this example, PN643 (full-length PN73 minus a C-terminal label),
PN690 (full-length PN73 with a C-terminal FITC-label) and PN708 (15-mer
derived by deletion of the 21 N-terminal residues of PN73) were tested at
5 μM, 10 μM, 20 μM and 40 μM. PN643 and PN690 were also
tested at 2.5 μM and PN690 was additionally tested at 1.25 μM.
PN643 and PN708 were also both tested at 80 μM.

[0331]As shown in Table 21, the non-FITC labeled PN73 (PN643) peptide
achieved nearly 100% uptake of siRNA at 10 μM concentration. However,
when the PN73 peptide was labeled with the FITC tag (PN690), its maximum
cell-uptake activity was reduced to approximately 70%. PN708 showed a
dose dependent increase in siRNA cell-uptake activity. PN708 achieved a
maximum siRNA cell-uptake activity of 95% at 80 μM. For the
full-length PN73 peptides, cell viability decreased as the concentration
of peptide increased. In contrast, cells incubated with the PN708 peptide
maintained over 90% cell viability in the presence of all tested
concentrations. In this example, the truncated peptide PN708 about
doubled the amount of Cy5-siRNA delivered into cells compared to the
full-length PN73 (PN690) peptide.

[0332]Polypeptide PN708 was characterized by determining its affect on
siRNA mediated target gene expression reduction. The C-terminal
FITC-label of the PN708 peptide was removed prior to assessing its
ability to enhance targeted gene expression reduction when complexed with
a siRNA. In the absence of the FITC-label, the truncated exemplary
polynucleotide delivery-enhancing polypeptide was named PN766 (refer to
Table 19 in Example 12). The ability of siRNA/peptide complexes to
modulate expression of the human tumor necrosis factor-α
(hTNF-α) gene was assessed (protocol details can be found in
Example 3). In this example, the random siRNA sequence, Qneg, served as a
negative control and the siRNAs LC20 and LC17 were used to target the
hTNF-α mRNA in human monocytes. The molar ratios of siRNA to
peptide tested were 1:5; 1:10; 1:25; 1:50; 1:75 and 1:100. Both LC20 and
LC17 were used at 20 nM concentration.

[0333]The knockdown results were that both the LC20/PN766 and LC17/PN766
siRNA/peptide complexes at 1:5; 1:10; and 1:25 reduced hTNF-α mRNA
levels to approximately 70%-80% of the Qneg siRNA negative control (i.e.,
20%-30% reduction in mRNA levels compared to the Qneg negative control).
The siRNA/peptide ratios of 1:50; 1:75 and 1:100 had no significant
affect on hTNF-α mRNA levels compared to the Qneg control. No
cytotoxicity effects were observed with human monocytes in the presence
of the PN766 peptide.

Example 16

Peptide Mediated siRNA Cell-Uptake Activity

[0334]The siRNA cell-uptake assay and MFI measurements were performed as
described previously in Examples 2 and 3. The data is summarized in Table
22. Each peptide was tested at 0.63 μM, 1.25 μM, 2.5 μM and 5
μM concentrations.

[0336]The siRNA cell-uptake activity for the polynucleotide
delivery-enhancing polypeptides listed in Table 23 complexed with siRNA.
Table 24 summarizes the siRNA cell-uptake data, mean fluorescence
intensity (MFI) measurements and cell viability data for each of the
polypeptides. Polypeptides that achieved a percent siRNA cell-uptake of
75% or greater are highlighted in grey in the "Treatment" column. The
specific percent siRNA cell-uptake for each these highlighted
siRNA/peptide complexes is also highlighted in grey in the "% siRNA
Cell-Uptake" column.

[0337]LC20 is an oligo used for the siRNA targeting of the human tumor
necrosis factor-alpha (hTNF-α) mRNA and is represented by the
ribonucleotide sequence:

TABLE-US-00032
UAGGGUCGGAACCCAAGCUUA (SEQ ID NO: 96)

[0338]siRNA uptake by cells was assessed by flow cytometry (refer to
Example 2 for details). Uptake was expressed as a percentage calculated
by dividing the number of cells containing Cy5-conjugated siRNA by the
total number of transfected and untransfected cells in culture. Mean
Fluorescence Intensity (MFI) was measured by flow cytometry and
determined the amount of Cy5-conjugated siRNA found within cells. The MFI
value directly correlates with the amount of Cy5-conjugated siRNA within
the cell, thus, a higher MFI value indicates a greater number of
Cy5-conjugated siRNA within the cells.

[0339]The data show that PN680, PN681, PN709, PN760, PN759, and PN682,
when complexed with siRNA, deliver siRNA into cells. The results for the
screening of polypeptides shown in Table 23 are shown in Table 24.

[0341]The polypeptides were further characterized for their ability to
transfect siRNAs into cells by analyzing Mean Fluorescence Intensity
(MFI). While the cell-uptake assay determined the percentage of cells
that contain the Cy5-conjugated siRNA, the MFI measurement determined the
relative mean quantity of Cy5-conjugated siRNA that entered the cells. As
shown in the column entitled "siRNA Cy5 MFI" of Table 24, delivery of the
Cy5-conjugated siRNA by the positive control peptide PN643 achieved a MFI
of approximately seven units. As expected, the "no treatment" negative
control has no measurable MFI. The polynucleotide delivery-enhancing
polypeptide PN665 was not tested by MFI. PN743, PN694 and PN714 had MFI
measurements significantly lower than that of the positive control. The
polynucleotide delivery-enhancing polypeptides PN680, PN709 and PN682
exhibited MFI measurements comparable to that of the PN643 positive
control while PN681 had an MFI double that of the positive control. PN760
and PN759 had MFI measurements that were approximately 13-fold and 6-fold
greater, respectively, than that of the positive control.

[0342]The following protocol was used to test the polynucleotide
delivery-enhancing polypeptides listed in Table 23. Approximately 80,000
mouse tail fibroblast (MTF) cells were plated per well in 24-well plates
the day before transfection in complete media. Each delivery peptide,
except the positive control, was tested at 0.63 μM, 2.5 μM, 10
μM and 40 μM concentrations in the presence of 0.5 μM
Cy5-conjugated siRNA. For siRNA/peptide complexes, the Cy5-conjugated
siRNA and peptide were diluted separately in Opti-MEM® media
(Invitrogen) at two-fold the final concentration. Equal volumes of siRNA
and peptide were mixed and allowed to complex five minutes at room
temperature. The siRNA/peptide complexes were added to cells previously
washed with phosphate buffered saline (PBS). Cells were transfected for
three hours at 37° C., 5% CO2. Cells were washed with PBS,
treated with trypsin, and then analyzed by flow cytometry. siRNA
cell-uptake was measured by the intensity of intracellular Cy5
fluorescence. Cell viability was determined using propidium iodide uptake
or AnnexinV-PE (BD Biosciences) staining. In order to differentiate the
cellular uptake from the membrane insertion of labeled siRNA (or
fluorescein-labeled peptide), trypan blue was used to quench any
fluorescence on the cell membrane surface. Trypan blue (Sigma) was added
to cells to a final concentration of 0.04% and re-run on the flow
cytometer to assess whether there was any change in fluorescence
intensity which would indicate fluorescence localized to the cell
membrane.

[0344]Human monocytes were used as a model system to determine the effect
of siRNA/peptide complexes on hTNF-α gene expression. Qneg
represents a random siRNA sequence and functioned as the negative
control. The observed Qneg knockdown activity was normalized to 100%
(100% gene expression levels) and the knockdown activity for each of the
following siRNAs A19S21 MD8, 21/21 MD8 and LC20 was presented as a
relative percentage of the negative control. A19S21 MD8, 21/21 MD8 and
LC20 are siRNAs that target hTNF-α mRNA.

[0345]The polypeptide PN602 is an acetylated form of the positive control
used in prior Examples and was used in this example as a positive control
for both the effective delivery of siRNA into human monocytes and the
permissive knockdown activity of hTNF-α mRNA levels mediated by
siRNA.

[0346]The data show that the polynucleotide delivery-enhancing polypeptide
PN680 delivers siRNAs into cells and permits effective siRNA mediated
gene silencing. The knockdown activity of PN602, PN680, and PN681 is
shown in Table 25. A "+" symbol indicates that the peptide/siRNA complex
had knockdown activity of 80% of the Qneg negative control siRNA (20%
reduction in mRNA levels compared to the Qneg negative control). A "+/-"
indicates that the peptide/siRNA complex had a knockdown activity of
approximately 90% of the Qneg negative control siRNA (10% reduction in
mRNA levels compared to the Qneg negative control). Finally, a "-"
indicates that the peptide/siRNA complex had no significant knockdown
activity compared to the Qneg negative control.

[0347]The results shown in Table 25 indicate that all three siRNAs
complexed with the positive control PN602 polynucleotide
delivery-enhancing polypeptide at ratios of 1:5 and 1:10 moderately
reduced hTNF-α gene expression levels compared to the Qneg negative
control complexed with the same polypeptide. However, the same siRNAs
complexed with the polynucleotide delivery-enhancing polypeptide PN681 at
1:5 and 1:10 showed little to no knockdown activity relative to the Qneg
negative control siRNA/PN681 complex. In contrast, the polynucleotide
delivery-enhancing polypeptide PN680 complexed with any of the
hTNF-α specific siRNAs at a 1:5 ratio exhibited significant
knockdown activity of the hTNF-α mRNA relative to the Qneg/PN680
control complex. Furthermore, the LC20/PN680 complex at a 1:10 ratio also
demonstrated significant knockdown activity compared to the Qneg/PN680
control complex.

[0348]Healthy human blood was purchased from Golden West Biologicals (CA),
the peripheral blood mononuclear cells (PBMC) were purified from the
blood using Ficoll-Pague plus (Amersham) gradient. Human monocytes were
then purified from the PBMCs fraction using magnetic microbeads from
Miltenyi Biotech. Isolated human monocytes were resuspended in IMDM
supplemented with 4 mM glutamine, 10% FBS, 1× non-essential amino
acid and 1× pen-strep, and stored at 4 C until use.

[0349]In a 96 well flat bottom plate, human monocytes were seeded at
100K/well/100 μl in OptiMEM medium (Invitrogen). The polynucleotide
delivery-enhancing polypeptides were mixed with 20 nM siRNA at a molar
ratio of 1:5 or 1:10 in OptiMEM medium at room temperature for five
minutes. At the end of incubation, FBS was added to the mixture (final
3%), and 50 μl of the mixture was added to the cells. The cells were
incubated at 37° C. for 3 hours. After incubation, the cells were
transferred to V-bottom plate and pelleted at 1500 rpm for five minutes.
The cells were resuspended in growth medium (IMDM with glutamine,
non-essential amino acid, and pen-strep). After an overnight incubation,
the monocytes were stimulated by application of LPS (Sigma) at 1 ng/ml
for three hours to increase expression of TNF-α expression levels.
After induction by LPS, cells were collected as above for mRNA
quantification, and supernatant was saved for protein quantification if
desired.

[0350]For mRNA measurement, branch DNA technology from Genospectra (CA)
was used according to manufacturer's specification. To quantitate mRNA
level in the cells, both house keeping gene (cypB) and target gene
(TNF-α) mRNA were measured, and the reading for TNF-α was
normalized with cypB to obtain relative luminescence unit.